Stimulus responsive polymers for the purification of biomolecules

ABSTRACT

The present invention provides novel and improved stimulus responsive polymers and methods of using the same for the purification of biomolecules.

PRIORITY DATA

The present application claims the benefit of priority of U.S.Provisional Patent Application No. 61/395,769, filed on May 17, 2010,the entire content of which is incorporated by reference herein in itsentirety.

FIELD OF INVENTION

The present invention relates to polymers useful for proteinpurification. In particular, the present invention relates, at least inpart, to stimulus responsive polymers useful for the purification of atarget molecule from a sample containing the target molecule and one andmore impurities.

BACKGROUND

Efficient and economic large scale purification of biomolecules such as,e.g., therapeutic proteins including antibodies, is an increasinglyimportant consideration for the biotechnology and pharmaceuticalindustries. Generally, the purification processes are quite elaborateand expensive and include many different steps. For example, typically,in the case of proteins, proteins are produced using cell culturemethods, e.g., using either mammalian or bacterial cell lines engineeredto produce the protein of interest by insertion of a recombinant plasmidcontaining the gene encoding that protein. In general, following theexpression of the target protein, its separation from one or moreundesired components including, e.g., host cell proteins, mediaby-products and DNA, poses a formidable challenge. Such separation isespecially important when the therapeutic proteins are meant for use inhumans and have to be approved by the Food and Drug Administration(FDA).

In general, separation and/or purification processes that are currentlybeing used for proteins, include at least the following steps: celllysis to recover an intracellular protein or recovery of a protein fromthe media in case of a secreted protein; removal of cells and cellulardebris using differential centrifugation or filtration to obtain aclarified sample containing the protein of interest; and use of avariety of chromatography media in a multi-step process to separate aprotein of interest from the various impurities in the sample.

Various types of polymers, including polyelectrolytes, have beenemployed in one or more steps for the purification of biomolecules,especially proteins. For example, the use of polyelectrolytes inflocculation to purify proteins is well established (see, e.g.,International PCT Patent Application No. WO2008/091740). This can beaccomplished with a wide range of polymers, with the only requiredgeneral characteristic being the polymer must have some level ofinteraction with a species of interest (e.g., a target molecule or animpurity). The most common methodology is the use of polymers containingion species, such as polyelectrolytes. Generally, polyelectrolytes areadded to the protein mixture and purification is achieved via selectiveflocculation of one or more components of the mixture. A criticaldrawback of this approach is that carefully controlled levels ofpolyelectrolytes have to be added in order to avoid residual polymercontamination (e.g., when polymer level too high) or inefficientflocculation (e.g., when polymer level too low). Because ion exchangeand other charged chromatography media are commonly used in thepurification of proteins, residual polyelectrolytes can potentially bindto the media used in downstream purification steps, thereby fouling andcomplicating the process.

Recently, technology has been developed which overcomes sonic of thechallenges associated with the use of polymers for purification ofbiomolecules (see, e.g., International PCT Publication No. WO2008/079302 A2). For example, stimulus-responsive or “Smart” polymershave been developed which can bind to both soluble (e.g., host cellproteins, DNA, cell culture additives) as well as insoluble (e.g., cellsand cellular debris) components (see, e.g., US Publication Nos.20080255027 and 20090036651). Although stimulus-responsive polymers showgreat promise in general. a key challenge that faces a broad use of suchpolymers is the existence of a simple stimulus which may be implementedat a variety of scales, ranging from laboratory scale to largeproduction scale.

SUMMARY OF THE INVENTION

The present invention provides novel polyelectrolyte based stimulusresponsive polymers which are easily scalable and operate over a widerange of pH and conductivity, thereby enabling their use in thepurification of a wide array of biomolecules including, e.g.,therapeutic proteins.

In some embodiments according to the present invention, a stimulusresponsive polymer comprising a polyelectrolyte backbone comprising oneor more hydrophobic groups is provided, where the polymer is capable ofbinding and precipitating a biomolecule of interest in a samplefollowing the addition of a stimulus.

In some embodiments, the polyelectrolyte backbone of a polymer accordingto the present invention comprises at least two monomeric units or atleast three monomeric units. In some embodiments, at least 50% of themonomeric units comprise a charge. In other embodiments, each monomericunit of the polyelectrolyte backbone comprises a charge.

In some embodiments, a stimulus responsive polymer according to thepresent invention comprises a polyamine backbone. In some embodiments,one or more hydrophobic groups is a phenyl group.

The stimulus responsive polymers according to the present invention areuseful for purifying a desired target molecule and do so by separatingthe desired target molecule from one or more undesirable entitiespresent in a sample along with the desired target molecule.

Accordingly, in some embodiments, a stimulus responsive polymeraccording-to the present invention binds and ‘precipitates a biomoleculeof interest, which itself is the desirable target molecule bound andprecipitated by the stimulus responsive polymer. In other embodiments, astimulus responsive polymer binds and precipitates a biomolecule ofinterest, which is an undesirable entity present in a sample along withthe desirable target molecule.

In some embodiments, the biomolecule of interest is a therapeuticpolypeptide (i.e., the desirable target molecule). In some embodiments,the therapeutic polypeptide is an antibody (e.g., a monoclonalantibody).

In other embodiments, the biomolecule of interest is selected from thegroup consisting of host cell protein, DNA, RNA, lipids, viruses,endotoxins, cell culture additives, whole cells and cellular debris.

In some embodiments, a polymer according to the present invention isresponsive to a stimulus which is a complex forming salt.

Also encompassed by the present invention are methods of using thepolymers described herein. The stimulus responsive polymers are uniqueand inventive over the polymers described in the prior art, in that theysupplant or improve upon one or more steps in a purification process,thereby to substantially increase the overall purity of a targetmolecule desired to be purified or separated from one or moreundesirable entities.

Accordingly, in some embodiments, a method of increasing the purity of atarget molecule is provided, wherein the method comprises the steps of(a) providing a sample comprising a target molecule and one or moreimpurities; (b) contacting the sample with a stimulus responsive polymercomprising a polyelectrolyte backbone comprising one or more hydrophobicgroups attached to the backbone under a first set of conditions suitablefor the polymer to bind the target molecule in solution. thereby to forma complex of polymer and the target molecule; and (c) adding a stimulusto the sample under a second set of conditions suitable to precipitatethe complex out of solution, where the precipitation of the complexresults in the separation of the target molecule from one or moreimpurities, thereby to increase the purity of the target molecule.

In some embodiments according to the methods of the present invention,the method further comprises the step of recovering a target moleculefrom the complex.

In another embodiment, a stimulus responsive polymer according to thepresent invention binds and precipitates one or more impurities, insteadof the target molecule, where the precipitation of a complex of thepolymer and one or more impurities results in the separation of thetarget molecule from the one or more impurities, thereby to increase thepurity of the target molecule. Accordingly, such a method comprises thesteps of: (a) providing a sample comprising a target molecule and one ormore impurities; (b) contacting the sample with a stimulus responsivepolymer comprising a polyelectrolyte backbone comprising one or morehydrophobic groups attached to the backbone under a first set ofconditions suitable for the polymer to bind the one or more impurities,thereby to form a complex of polymer and the one or more impurities; and(c) adding a stimulus to the sample under a second set of conditionssuitable to precipitate the complex, where the precipitation of thecomplex results in the separation of the target molecule from one ormore impurities, thereby to increase the purity of the target molecule.

In some embodiments, a stimulus responsive polymer according to thepresent invention comprises the following structure:

where x and y represent monomeric units of the polymer; R₁ and R₂ arecharged groups which form a part of the polyelectrolyte backbone (B);and R₃ is a hydrophobic group attached to a charged group in thebackbone. The ratio of y monomeric units (i.e., having a hydrophobicgroup attached to the backbone) to the total number of monomeric units(i.e., sum of x and y monomeric units) represents the “percent ofhydrophobic modification” of the polymer.

In some embodiments, a stimulus responsive polymer according to thepresent invention comprises the following structure:

where x, y and z arc monomeric units in the polymer; R₁, R₂ and R₃ arecharged groups which form a part of the polyelectrolyte backbone (B); R4is a hydrophobic group attached to a charged group in the backbone; andR₅ is a functional group attached to a charged group in the backbone.The ratio of y monomeric units (i.e., having hydrophobic group attachedto backbone) to the total number of monomeric units in the polymer(i.e., sum of x, y and z monomeric units) represents the “percenthydrophobic modification” of the polymer. Further, the ratio of zmonomeric units (i.e., having a functional group attached to a chargedgroup on the backbone) to the total number of monomeric units (i.e., sumof x, y and z units) represents the “percent functional groupmodification” of the polymer.

In general, it is understood that a polymer encompassed by the presentinvention may have “n” number of any of monomeric units x, y or z,described herein, where n is equal or greater than two.

In still other embodiments, a stimulus responsive polymer according tothe present invention comprises the following structure:

where x and y represent monomeric units; R₁, R₂ are aliphatic amine,groups (e.g., primary or secondary and/or aromatic amines) which form apart of a carbon containing backbone of a polyelectrolyte; and R₃ is ahydrophobic group attached to the amine group R₂ and contains 4 or morecarbon atoms (e.g., an alkyl group, an alkenyl group, an aralkenyl groupor a fluorocarbon group). In some embodiments, the ratio of y (i.e.,monomeric units having a hydrophobic group attached to a charged groupin the polyelectrolyte backbone) to x (i.e., unmodified charged group inthe polyelectrolyte backbone) is 0.01 to 0.75 or 0.05 to 0.75.Accordingly, the percent hydrophobic group modification would be between1% to 75% or between 5% to 75% of the total polyelectrolyte monomericunits (i.e., x+y).

in yet other embodiments, a stimulus responsive polymer according to thepresent invention comprises the following structure:

where R₁, R₂ and R₃ are aliphatic amine groups which form a part of thecarbon containing polyelectrolyte backbone (e.g., primary or secondaryamines and/or aromatic amines); R₄ is a hydrophobic group containing 4or more carbon atoms and selected from alkenyl, aralkyl and aralkenylgroups; and R₅ is a hydrophobic group containing 4 or more 4 carbonatoms and selected from an alkyl or a fluorocarbon group. The ratio of ymonomeric units to the total number of polyelectrolyte monomeric unitsis between 0.01 and 0.75. The ratio of z monomeric units to the totalnumber of polyelectrolyte monomeric units is between 0.05 and 0.5 orbetween 0.01 and 0.5. Accordingly, the percent hydrophobic groupmodification is between 1% and 75% or between 5% and 75% and the percentfunctional group modification is between 1% and 50% or between 5% and50%.

Additional methods of using the stimulus responsive polymers accordingto the present invention include methods which enable the purificationof a target molecule or product of interest (e.g., an antibody) whileminimizing the amount of residual polymer in the sample.

In some embodiments, a method of separating a target molecule (e.g., anantibody) from one or more impurities using a stimulus responsivepolymer according to the present invention while minimizing residualamounts of polymer is provided, where the method comprises the steps of:(a) providing a sample comprising a target molecule and one or moreimpurities; (b) contacting the sample with a stimulus responsive polymerunder a first set of conditions suitable for the polymer to bind the oneor more impurities, thereby to form a first complex of polymer and oneor more impurities, where the first set of conditions comprise adjustingpH or salt concentration of the sample before or after the addition ofthe polymer; (c) precipitating the first complex from the sample under asecond set of conditions; (d) contacting the sample with a multivalention, thereby to form a second complex of residual polymer andmultivalent ion; (e) precipitating the second complex; and (f)recovering the target molecule from the sample; thereby to separate thetarget molecule from one or more impurities in the sample while reducingthe amount of residual polymer in the sample.

In some embodiments, the target molecule is an antibody. In a particularembodiment, the antibody is a monoclonal antibody.

In a particular embodiment, recovering the target molecule in variousmethods according to the present invention comprises a chromatographystep. In another embodiment, recovering the target molecule comprises afiltration step.

In some embodiments, methods according to the present invention mayinclude two or more steps which employ a stimulus responsive polymeraccording to the invention. For example, a stimulus responsive polymermay be used to precipitate one or more impurities in one step of thepurification process and same or different polymer may be used toprecipitate a target molecule or the desired product in a different stepof the process.

In some embodiments, the one or more impurities is selected from thegroup consisting of host cell protein, DNA, RNA, antibody aggregates,viruses, endotoxins, whole cells, cellular debris and cell cultureadditives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the reaction of a polyallylamine polymerwith benzylchloride

FIG. 2 depicts the reaction scheme for hexanoic acid and tert-butylmodified polyallylamine (HC-t-BuMPAA)

FIG. 3 is a graph depicting the effect of sodium chloride on themultivalent ion stimulus and pH responsiveness of hexanoic acid andtert-butyl modified polyallylamine (HC-t-BuMPAA). The X-axis depicts pHand Y-axis depicts turbidity of the centrate (i.e., output of acentrifuge).

FIG. 4 depicts the synthesis of polyvinylamine, as described in Example29.

FIG. 5 depicts a reaction for the deprotection of polyamine afterpolymerization.

FIG. 6 depicts a reaction of polyvinylamine with benzylchloride

FIG. 7 depicts a polymerization and reaction scheme to form amultivalent ion stimulus responsive copolymer.

FIG. 8 depicts an NMR spectrum for modified polyvinylamine (PVA) fromExample 35. Integration of the 1H NMR shows benzyl modification level ofabout 18%.

FIG. 9 depicts an NMR spectrum for modified polyallylamine from Example36. Integration of the I H NMR shows benzyl modification level of about33%

FIG. 10 depicts a graph demonstrating the effect of polymer dosing for anon-stimulus responsive polymer (e.g., chitosan) and a stimulusresponsive polymer (e.g., benzyl modified polyallylamine) on centrateturbidity. The X-axis is the polymer dose (wt %) and the Y-axis is thecentrate turbidity (NTU), as described in Example 37.

FIG. 11 depicts a graph demonstrating the effect of polymer dose in thepresence and absence of a stimulus for a benzyl polyallylamine stimulusresponsive polymer. The X-axis is the polymer dose in wt% and Y-axis isthe centrate turbidity (NTU), as described in Example 38.

FIG. 12 depicts a typical scheme used for the purification ofbiomolecules.

FIG. 13 depicts a purification scheme including a stimulus responsivepolymer used to improve the clarification of the cell culture. Thestimulus responsive polymer removes one or more impurities, however, thepolymer does not bind the desired target molecule.

FIG. 14 depicts a purification scheme including a stimulus responsivepolymer used to improve the clarification of the cell culture. Thestimulus responsive polymer removes one or more impurities viaflocculation, however, the polymer does not bind the desired targetmolecule and the residual polymer is removed by adding a stimulus afterclarification.

FIG. 15 depicts a purification scheme including a stimulus responsivepolymer used to improve the clarification of the cell culture. Thestimulus responsive polymer removes one or more impurities, however, thepolymer does not bind the target molecule and the residual polymer isremoved by an additional adsorptive filtration step followingclarification.

DETAILED DESCRIPTION

The present invention provides, at least in part, a novel and improvedstimulus responsive polymer comprising a polyelectrolyte backbonemodified with one or more hydrophobic groups, where the polymersolubility can be altered by the addition of a stimulus.

The stimulus responsive polymers and methods of using the same describedherein are more efficient than those described in the prior art, inthat, they provide an improved range of pH for purification of desiredtarget molecules, including, e.g., proteins, as well as removal ofundesirable entities such as impurities, e.g., host cell proteins, DNA,RNA, lipids, endotoxins, cell culture additives, cells and cellulardebris. In some embodiments, the polymers described herein areresponsive to low concentrations of simple multivalent salts, therebyallowing for improved scalability and reduced conductivity relative toexisting salt responsive polymers. In various embodiments, the polymersaccording to the present invention can effectively remove whole cells,cellular debris as well as other soluble impurities from cell culturemedia. The polymers can also effectively remove impurities from proteinmixtures containing a protein of interest and one or more impurities.Further, various polymers described herein are able to effectivelycapture target molecules and proteins/products of interest in a sample,thereby to separate them from one or more impurities present in thesample and increase the purity of the target molecule.

Also encompassed by the present invention are methods of using thepolymers described herein for purification of target molecules, e.g.,therapeutic proteins, using a wide array of conditions.

Without wishing to be bound by theory, it is contemplated that thestimulus responsive polymers described herein can be used for thebinding and precipitation of either a desired target molecule, e.g., atherapeutic protein or a desirable product, or an undesired entity,e.g., one or more impurities including, e.g., host cell protein, DNA,RNA, lipids, endotoxins, cell culture additives, whole cells andcellular debris. In general, a molecule bound by a polymer according tothe present invention is referred to as a biomolecule of interest,whether it is the desired target molecule or an undesired entity.

The selection of a particular stimulus responsive polymer to use, asdescribed herein, is determined based on what the polymer is intended tobind. For example, in case of a biomolecule of interest which possessesa net negative charge at a pH above its pI (e.g., whole cells, cellulardebris, DNA, endotoxins, and proteins), a stimulus responsive polymercomprising a polyelectrolyte backbone which is cationic (i.e.,positively charged) is desirable to use. On the other hand, in case of abiomolecule of interest which comprises a net positive charge at a pHbelow its pI (eg. proteins), a stimulus responsive polymer comprising apolyelectrolyte backbone which is anionic (i.e., negatively charged) isdesirable to use.

A positive charge could be inherent to the polymer under conditions usedduring the purification process or, the positive charge can be generatedwith a change in pH which renders the stimulus responsive polymercharged.

An important parameter affecting the overall recovery of a biomoleculeis the ratio of hydrophobic modification groups to the remainingunmodified charged groups in the polyelectrolyte backbone. For example,as the percentage of hydrophobic groups increases so does the resultingloss of the biomolecule through non-specific interactions. Therefore,for a given biomolecule, a specific ratio of charged groups tohydrophobic groups can be used in order to maximize biomoleculerecovery. Additionally, a high percentage of hydrophobic groups maylimit polymer solubility and the effectiveness of charged groups on thepolyelectrolyte backbone.

Further, modification of a charged amine group in the backbone of thepolyelectrolyte polyallylamine with benzyl chloride, results in asecondary amine which is charged under a wide array of pH conditions.However, such a benzyl modification adds a steric bulk to the aminegroup which can affect charge-charge interactions. Additionally,modification of a charged group in a polyelectrolyte backbone with ahydrophobic group can result in a reduction in the number of chargedgroups. For example, modification of an amine group of polyallylaminewith benzyl chloride results in the formation of an amide linkage whichis not a charged group, thereby resulting in a reduction in the numberof charged groups in the backbone. Accordingly, a reduction in thenumber of charged groups can also affect both the polymer's solubilityas well as the polymer's ability to bind through charge-chargeinteractions.

While, certain polymers according to the present invention are cationicand others are anionic, hybrid polymers may also be synthesized, whichcomprise a polyelectrolyte backbone which is cationic and modified withone or more hydrophobic as well as anionic groups. In case of suchhybrid polymers, unmodified groups on the cationic polyelectrolyte areresponsive to a complex forming salt, while the anionic modificationgroups on the backbone can bind a biomolecule of interest whichpossesses a net positive charge. Accordingly, the ratio of unmodifiedcationic groups in the polyelectrolyte backbone to anionic andhydrophobic groups is important to determine the solubility and stimulusnecessary for the polymer to complex and capture the biomolecule ofinterest. For example, too few unmodified cationic groups in thepolyelectrolyte backbone can result in limited to no response tostimulus. Whereas, too few anionic groups can limit the ability tocapture a biomolecule of interest.

The requisite level of modification of polyelectrolyte backbone byhydrophobic groups as well as the type and amount of stimulus that isused can be determined based on the biomolecule of interest to bepurified using the polymer as well as the conditions used, and theinherent solubility and molecular weight of the polymer backbone.. Forexample, in order to minimize the amount of stimulus used, e.g., amultivalent salt, it is desirable to have more hydrophobic groupsattached to the polyelectrolyte backbone. Alternatively, increasedamount of multivalent ion stimulus reduces the extent of the hydrophobicmodification or percent hydrophobic modification required. In someinstances, it may be possible to completely eliminate the need forhydrophobic modification, for example, if the multivalent ion stimulusis at a very high concentration. Alternatively, increasing the polymermolecular weight reduces the inherent solubility of the polyelectrolytebackbone and may allow for low (5% or less) or no polyelectrolytehydrophobic modification. However, the elimination of hydrophobicmodification can increase the amount of residual polymer for lowermolecular weight or more soluble polymer backbones at high polymerdoses.

In various embodiments, the extent of hydrophobic modification on apolyelectrolyte backbone ranges from 1% to 85% or 5% to 50%.Accordingly, depending on the biomolecule of interest to be bound by astimulus responsive polymer, the percentage of hydrophobic modificationis at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20′Y0, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%.

In some embodiments, a complex forming salt is used as a stimulus. Invarious embodiments, the concentration of the complex forming saltranges from 2 mM to 500 mM, or from 25 mM to 100 mM. Exemplary complexforming salts include but are not limited to multivalent ions such as,e.g., citrate, phosphate, sulfate and EDTA, and ion-association saltssuch as perchlorate, dodecyl sulfate sodium salt, dodecyl benzenesulfate, Fe(II)-4-chloro-2-nitrophenol anion, tetraphenyl borate sodiumsalt and hexanitrodiphenol amine (see. e.g., ANALYTICAL SCIENCES,DECEMBER 1987, VOL. 3, p. 479). In general, an ordinary skill in the artwould be familiar with numerous complex forming salts which are known inthe art and may be used as a stimulus for the polymers described herein.

The amount of a complex forming salt which is required to induceprecipitation depends on factors such as, e.g., pH, polymerconcentration and concentration of biomolecule of interest in a sample.For example, some polyelectrolytes such as polyallylamine have a chargedensity which varies with pH (level of amine protonation). As the pH isincreased, the level of charge density is reduced so the degree ofstimulus required to induce precipitation will be different than at alower pH or higher charge density state.

In general, a stimulus responsive polymer according to the presentinvention maybe added to a feedstock containing a target molecule or atarget molecule containing sample, in the form of a solid or in the formof a liquid. The final polymer concentration is generally between 0.01%and 2%. In some methods described herein, a mixture of a polymer and abiomolecule of interest is generated followed by the addition of astimulus, e.g., a complex forming salt such as a multivalent anion. Theamount of stimulus may depend on the polymer concentration. For example,a polymer concentration of 2% will require a higher amount of stimulusrequired to induce polymer precipitation. It is important that thestimulus is applied in the correct or in a slight excess amount in orderto ensure complete polymer precipitation through the stimulus response.This is in contrast to polymer flocculation, where overdosing leads toproblematic residual polymer.

The present invention can be used in a variety of purification schemes.The stimulus responsive polymer can be beneficial at any step of theprocess, although the preferred use is at the beginning of the processduring clarification or during capture of the target molecule. A singlestimulus responsive polymer or mixture of polymers can be added in oneor more steps and subsequently precipitated using one or more stimuli.The stimulus can be applied before, during or after the polymerassociates with the biomolecule of interest (i.e., one or moreimpurities or a desired target molecule). Also, the stimulus can beapplied before, during or after the removal of the precipitate, which isgenerally in solid form. The precipitate can be subsequently removedusing one or more techniques known in the art and/or those describedherein such as, e.g., filtration, settling, centrifugation or any othersolid/liquid separation method or a combination of methods insimultaneous, parallel or series separation schemes.

Addition of the stimulus responsive polymer can be accomplished inseveral ways. The cell culture media may be adjusted to a desiredcondition prior to the addition of a stimulus responsive polymer, forexample adjustment (e.g., reduction) of pH and/or conductivity. Thestimulus responsive polymer can then be added to the cell culture mediaand mixed. The stimulus responsive polymer can be added in a liquid or asolid format. The polymer containing solution itself can be formulatedjust that it adjusts the pH of the cell culture media to a desiredcondition. For example, the stimulus responsive polymer can be dissolvedin a concentrated acetic acid solution. The concentration of this aceticacid solution can be altered based on the volume, fermentation solutioncondition and protein concentration in order to provide the necessary pHadjustment upon addition of the stimulus responsive polymer, thereby toresult in the desired polymer concentration and solution pH. Thestimulus responsive polymer can be added in concentrations wherespontaneous flocculation occurs, typically in the range of 0.01 to 0.1%wt polymer or 0.01 to 0.5% wt polymer, depending on the type and percentsolids, such that the solution becomes cloudy and begins to form aprecipitate. Alternatively, the stimulus responsive polymer can be addedin concentrations where spontaneous flocculation does not occur butpolymer-biomolecule association does occur, e.g., typically, in therange of 0.5% to 2% wt polymer, and the solution could be clear orslightly cloudy or more turbid than The original solution. Also, thestimulus responsive polymer can be added in concentrations where amixture of spontaneous flocculation and polymer-biomolecule associationoccurs.

Although, use of a stimulus in a purification scheme is more desirableas it alleviates some of the problems associated with overdosing withthe polymer, e.g., as in case of flocculation processes, it iscontemplated that the polymers described herein may also be used asflocculants.

In an exemplary purification scheme, a stimulus responsive polymeraccording to the present invention is added to the cell culture afterfermentation is complete and polymer is formulated to bind a biomoleculeof interest which is not the desired target molecule. In such a method,a stimulus responsive polymer is added to the cell culture under a firstset of conditions, e.g., conditions which may be adjusted before, duringor after the addition of the polymer which binds the biomolecule ofinterest. After the stimulus responsive polymer is added under the firstset of conditions, a stimulus is added under a second set of conditions,thereby generating a precipitate which includes the biomolecule ofinterest (e.g., one or more impurities such as cells, cellular debris,host cell proteins, DNA, endotoxins, and viruses). The solid precipitatecan be subsequently removed by centrifugation and/or filtration, therebyresulting in a clarified cell culture fluid. The resulting clarifiedcell culture fluid may be subsequently passed through a capture stepusing a chromatography media to bind the desired target molecule. Thetarget molecule may subsequently be eluted from the capture step.Accordingly, in some cases, by using a stimulus responsive polymeraccording to the present invention, which enables removal of one or moreimpurities at the clarification step, number of additional steps may bereduced/eliminated or modified.

In some embodiments, a stimulus responsive polymer is added to the cellculture under conditions where the polymer binds a biomolecule ofinterest, which is not the target molecule. After the stimulusresponsive polymer is well mixed at the desired solution conditions, aflocculate which includes the biomolecule of interest is allowed toform. The solid containing the biomolecule of interest is removed byprimary clarification. The resulting cell culture fluid is collected anda stimulus is applied to precipitate residual polymer. The precipitatedresidual polymer is subsequently removed by secondary clarification. Theresulting clarified cell culture solution is passed through a capturestep using a chromatography media to bind the target molecule. It may bepossible to remove the residual polymer using the addition of a stimulusat any of the purification steps following primary clarification. Also,it may be possible to add a stimulus to remove residual polymer at anystep or add one or more stimuli multiple times throughout the process.

In another purification scheme, a stimulus responsive polymer is addedto the cell culture fluid after fermentation is complete underconditions suitable for the polymer to bind one or more impurities, suchthat the polymer does not bind the target molecule. After the stimulusresponsive polymer is well mixed under the desired solution conditions,a stimulus is added which forms a solid precipitate with one or moreimpurities. The solid precipitate is removed by centrifugation and/orfiltration. The resulting clarified cell culture solution is passedthrough a filter which is capable of binding the stimulus responsivepolymer. One of ordinary skill in the art can readily select/identify afilter which can bind the stimulus responsive polymer. For example, afilter with similar properties to the biomolecule of interest which isto be bound by the polymer may be provided. Alternatively, a filterpossessing a charged group that has the same charge as the biomoleculeof interest and a charge opposite that of the stimulus responsivepolymer may be used.

Membranes, packed beds or filters may also be used to remove a polymerfrom solution. For example, an anionic membrane containing sulfonic acidgroups could be used to remove a polyamine stimulus responsive polymer.Also, a filter with binding properties similar to the stimulus used toprecipitate the polymer can be used. If the stimulus is a multivalentanion, then providing a filter with a surface that contains multivalentanions can remove the polymer from solution. For example, a membranemodified with polyvinylphosphate could bind to polyamines in much thesame way that phosphate ions can complex with polyamines, thereby toinduce precipitation. Also, beads (e.g., polymethacrylate) modified withpolyvinylphosphate could be used. In some embodiments, followingfiltration of the solution with a filter that can remove the stimulusresponsive polymer, the resulting solution is passed through a capturestep using a chromatography media to bind the target biomolecule. It ispossible to remove the polymer using a chromatography media, depthfilter or other porous material that can bind the stimulus responsivepolymer. It is also possible to remove the polymer with adsorptive meansat a single step or more than one or multiple steps. Further, it ispossible to remove the polymer via adsorptive means at any step afterthe polymer's addition to the mixture.

It is anticipated that the present invention can be used in manyvariations of the purifications schemes described herein. The stimulusresponsive polymer can be used to substitute or enhance both theclarification and capture steps. For example, two separate stimulusresponsive polymers could be used; the first polymer which binds one ormore impurities but does not bind the target molecule and a secondpolymer which binds the target molecule. These two polymers could beapplied in separate steps or a single step. Likewise, a single polymercould be used which has functional groups capable of binding the targetmolecule and a polyelectrolyte backbone capable of binding one or moreimpurities. The single polymer could bind the target molecule and one ormore impurities in a single step or multiple steps. Subsequent elutionof the target molecule from the polymer may occur after severaldifferent precipitation/stimulus additions or washing steps. Thestimulus responsive polymer can be added after the capture step and usedto clarify suspended solids and impurities which result from virusinactivation or other steps after the capture step. The stimulusresponsive polymer can also substitute for or enhance polishing steps.

In some embodiments, a virus inactivation step (i.e., exposure ofsolution to low pH, surfactants or heat) is included. The solutionconditions may be adjusted and the sample processed through a series ofpolishing steps (i.e., one or more of ion exchange, hydrophobicinteraction, mixed mode and others). The solution may then undergo aseries of filtration steps, including virus filtration andultrafiltration or diafiltration.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the

DETAILED DESCRIPTION I. Definitions

The term “stimulus” or “stimuli,” as used interchangeably herein, ismeant to refer to a physical or chemical change in the environment whichresults in a response by a stimulus responsive polymer according to thepresent invention. Accordingly, the present invention provides novelpolymers which are responsive to a stimulus and which stimulus resultsin a change in the solubility of the polymer. Examples of stimuli towhich one or more polymers described herein are responsive, include, butare not limited to, e.g., changes in temperature, changes inconductivity and/or changes in pH. In some embodiments, a stimuluscomprises addition of a complexing agent or a complex forming salt to asample. In various embodiments, a stimulus is generally added after theaddition of a polymer to a sample. Although, the stimulus may also beadded during or before addition of a polymer to a sample.

The term “polymer” as used herein, refers to a molecule formed bycovalent linkage of two or more monomer units. These monomer units canbe synthetic or occur in nature. The polymers formed by the repeatingunits can be linear or branched. Examples of polymers include, but arenot limited to, polyethylene glycol, polypropylene glycol, polyethylene,polyallylamine, polyvinylalcohol, polystyrene and copolymers (e.g.polystyrene-co-polypyridine, polyacrylic acid-co-methyl methacrylate,pluronics, PF68 etc). In some embodiments according to the presentinvention, polymers comprise a polytelectrolyte backbone. Also describedherein are copolymers, which may be used in the methods according to thepresent invention, where the copolymers are responsive to a stimulus. Ingeneral, it is understood that in case of polymers, the monomeric unitsare of the same type, whereas, a copolymer will usually have differenttypes of monomeric units.

The term “stimulus responsive polymer,” as used herein, is a polymer orcopolymer which exhibits a change in a physical and/or chemical propertyafter the addition of a stimulus. A typical stimulus response is achange in the polymer's solubility. For example, the polymerpoly(N-isopropylacrylamide) is water soluble at temperatures below about35° C., but become insoluble in water at temperatures of about 35° C. Ina particular embodiment, a stimulus responsive polymer is apolyallylamine or a polyvinylamine polymer which is responsive to amultivalent ion stimulus (e.g, phosphate stimulus).

The term “polyelectrolyte backbone,” as used herein, refers to a carboncontaining polymer comprising two or more monomeric units, where atleast 50% of the units, or at least 55% of the units, or at least 60% ofthe units, or at least 65% of the units, or at least 70% of the units,or at least 75% of the units, or at least 80% of the units, or at least85% of the units, or at least 90% of the units, or at least 95% of theunits, contain a charged functionality. In other words, at least 50% ofthe monomeric units include a charged group which forms a part of theunit. In some embodiments, a polyelectrolyte backbone described hereincontains at least two or more monomeric units, where each of the unitscontains a charged functionality. In case of the polyelectrolytebackbone of polymers, in which each of the monomeric units contains acharged functionality, such polymers may be referred to as referred toas “continuous polyelectrolytes.” Exemplary polyelectrolytes include,but are not limited to, polyallylamine, polyvinylamine, polyacrylicacid, polyethyleneimine, chitosan, and polyvinylphosphoric acid. It isalso contemplated that one or more entities, which are different fromthe monomeric units, may be linked to polyelectrolyte backbone.

The term “hydrophobic group,” as used herein, refers to a nonpolarentity or chemical group, which has little to no affinity for water.Exemplary hydrophobic groups include, but are not limited to, phenylgroups, tertiary butyl groups, cyclic hydrocarbons, polycylic aliphatichydrocarbons, polycyclic aromatic hydrocarbons, and short chainhydrocarbons such as hexyl and octyl groups. In a particular embodiment,the hydrophobic group is a phenyl group. The hydrophobic group can alsobe a non-hydrocarbon and contain heteroatoms such as nitrogen, oxygen,sulfur, phosphorus etc. In various embodiments according to the presentinvention, stimulus responsive polymers are provided, which comprise apolyelectrolyte backbone having one or more hydrophobic group attachedto a charged group in the backbone. Without wishing to be bound bytheory, it is understood that the number of hydrophobic groups attachedto the polyelectrolyte backbone is important for altering the polymersolubility, thereby to improve the stimulus responsiveness of thepolymer. However, it may be undesirable to have a number of hydrophobicgroups which render the polymer water insoluble without a stimulus.

The percentage of charged groups in a polyelectrolyte backbone which aremodified with a hydrophobic group is generally referred to as “percenthydrophobic modification” of the polymer. Accordingly, in variousembodiments, the percent hydrophobic modification is important andranges 1% to 85% or from 5% to 85%. Accordingly, the percent hydrophobicmodification may be at least 1%,or at least 2%, or at least 3%, or atleast 4% or at least 5%, or at least 6%, or at least 7%, or at least 8%,or at least 9%, or at least 10%, or at least 15%, or at least 20%, or atleast 25%, or at least 30%, or at least 35%, or at least 40%, or atleast 45%, or at least 50%, or at least 55%, or at least 60%, or atleast 65%, or at least 70%, or at least 75%, or at least 80%, or atleast 85%.

The term “percent hydrophobic modification” as used herein generallyrefers the ratio of unmodified polyelectrolyte charged groups tohydrophobic group modified polyelectrolyte charged groups as a percentof the total polyelectrolyte monomeric units in a polyelectrolytepolymer backbone.

In some embodiments, a hydrophobic group attached to a polyelectrolytebackbone further has a charged group attached to the hydrophobic group,which is distinct entity from the charged group in the backbone.

As used herein, the term “alkyl,” generally refers to a straight orbranched hydrocarbon chain. Straight chain or branched chain hydrocarbonchains refer to any substituted or unsubstituted acycliccarbon-containing compounds, including, e.g., alkanes, alkenes andalkynes. Examples of alkyl groups include lower alkyl, for example,methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl oriso-hexyl; upper alkyl, for example, n-heptyl, n-octyl, iso-octyl,nonyl, decyl, and the like; lower alkylene, for example, ethylene,propylene, propylyne, butylene, butadiene, pentene, n-hexene oriso-hexene; and upper alkylene, for example, n-heptene, n-octene,iso-octene, nonene, decene and the like. An ordinary skill in the artwould be familiar with numerous straight, i.e., linear, as well asbranched alkyl groups, which are encompassed by the present invention.

In addition, such alkyl groups may also contain various substituents inwhich one or more hydrogen atoms is replaced by a functional group.Examples of functional groups include but not limited to, carboxylic,sulfonic, phosphonic groups and the like. As used herein, the term“alkenyl” refers to a straight or branched hydrocarbon chain, whereatleast one of the carbon-carbon linkages is a carbon-carbon double bond.

As used herein, the term “aralkyl” refers to an alkyl group which isterminally substituted with at least one aryl group.

As used herein, the term “aralkenyl” refers to an alkenyl group which isterminally substituted with at least one aryl group.

As used herein, the term “aryl” refers to a hydrocarbon ring bearing asystem of conjugated double bonds, often comprising at least six n (pi)electrons. Examples of aryl groups include, but are not limited to,phenyl, naphthyl, anisyl, toluyl and xylenyl.

As used herein, the term “fluorocarbon” refers to a straight or branchedcarbon chain in which one or more hydrogen atoms is replaced by afluorine group. Straight chain or branched chain fluorocarbon chaingenerally refers to any substituted or unsubstituted acycliccarbon-containing compound, including, e.g., alkanes, alkenes andalkynes. Examples of alkyl groups include lower alkyl, for example,methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl oriso-hexyl; upper alkyl, for example, n-heptyl, n-octyl, iso-octyl,nonyl, decyl, and the like; lower alkylene, for example, ethylene,propylene, propylyne, butylene, butadiene, pentene, n-hexene oriso-hexene; and upper alkylene, for example, n-heptene, n-octene,iso-octene, nonene, decene and the like. In general, it is understoodthat one or ordinary skill in the art would be familiar with numerousstraight, i.e., linear, as well as branched alkyl groups, which arewithin the scope of the present invention. In addition, such alkylgroups may also contain various substituents in which one or morehydrogen or one or more fluorine atoms is replaced by a functionalgroup. Examples of functional groups include, but are not limited to,carboxylic, sulfonic, phosphonic groups and the like.

The term “functional group,” as used herein, is a group which impartsadditional functionality to a polymer described herein. In other words,a functional group is a group, which is distinct from a hydrophobicgroup, and is also attached to the polyelectrolyte backbone, e.g., to acharged group in the backbone. For example, in some embodiments, afunctional group can be a ligand for changing the binding properties ofthe polymer, for example, carboxylic acid, sulfonic acid, sulfate,primary amine, quaternary amine and diethylamino groups. The functionalgroup can also alter properties or provide additional desired propertiesto the polymer such as, for example, altering a stimulus response ormaking the polymer responsive to a second stimulus. Exemplary functionalgroups for altering stimulus response behavior include, but are notlimited to, carboxylic acid group (pH responsive), pyridine group (pHresponsive) and N-isopropylacrylamido group (temperature response).

The term “ligand,” as used herein, generally refers to an entity whichprovides a specific binding capability for another entity. Examples of“ligands” include, but are not limited to, ion exchange groups,bioaffinity or biospecific groups, hydrophobic groups, thiophilicinteraction groups, chelate or chelating groups, groups having so calledpi-pi interactions with target compounds, hydrogen bonding groups, andhydrophilic groups.

The term “flocculation,” as used herein, refers to the addition of aflocculant, such as a polymer described herein, to a solution in orderto remove one or more suspended insoluble or soluble impurities. Thepolymer must be added to the solution at a concentration which allowsfor spontaneous formation of insoluble aggregates which can be removedfrom solution via typical solid-liquid separation methods.

The term “composition,” “solution” or “sample,” as used herein, refersto a mixture of a target molecule or a desired product to be purifiedusing one or more stimulus responsive polymers described herein alongwith one or more undesirable entities or impurities. In someembodiments, the sample comprises feedstock or cell culture media intowhich a target molecule or a desired product is secreted. In someembodiments, the sample comprises a target molecule (e.g., a therapeuticprotein or an antibody) along with one or more impurities (e.g., hostcell proteins, DNA, RNA, lipids, cell culture additives, cells andcellular debris). In some embodiments, the sample comprises a targetmolecule of interest which is secreted into the cell culture media. Insome embodiments, a sample from which a target molecule is to bepurified using one or more stimulus responsive polymers described hereinis “partially purified” prior to contacting the sample with a stimulusresponsive polymer. Partial purification may be accomplished, forexample, by subjecting the sample to one or more purification steps,such as, e.g., one or more non-affinity chromatography steps. The targetmolecule may be separated from one or more undesirable entities orimpurities either by precipitating the one or more impurities or byprecipitating the target molecule.

In some embodiments, a stimulus responsive polymer according to thepresent invention binds to a biomolecule of interest which itself is atarget molecule or product (e.g., a target protein or polypeptide),under a first set of conditions and precipitates the target moleculeunder a second set of conditions, e.g., upon the addition of a stimulusto the sample. In other embodiments, a biomolecule of interest is amolecule other than a target molecule. In other words, the biomoleculeof interest bound by a stimulus responsive polymer described herein maybe a molecule which is not desired to be associated with a targetmolecule in a sample. Without wishing to be bound by theory, it iscontemplated that in sonic embodiments, a stimulus responsive polymeraccording to the present invention binds and precipitates one or more ofhost cell proteins, DNA, whole cells, cellular debris, viruses,endotoxins, and/or cell culture additives, upon the addition of astimulus. Accordingly, a target molecule (e.g., a target protein orpolypeptide) could be purified using a polymer described herein eitherby precipitating the desired target molecule or by precipitating one ormore undesirable entities (e.g., one or more impurities) which may bepresent in a sample containing the desired target molecule.

The term “precipitate,” precipitating” or “precipitation,” as usedherein, refers to the alteration of a bound (e.g., in a complex with abiomolecule of interest) or unbound polymer from an aqueous and/orsoluble state to a non-aqueous and/or insoluble state.

The term “biomolecule of interest,” as used herein, refers to anymolecule which is bound and precipitated by a stimulus responsivepolymer described herein. For example, the biomolecule of interest canbe a desired target molecule such as, for example, a desired product orpolypeptide of interest (e.g., an antibody), or it can be an undesirableentity, which needs to be removed from a sample containing the desiredtarget molecule. Such undesirable entities include but are not limitedto, for example, one or more impurities selected from host cell protein,DNA, RNA, protein aggregates, cell culture additives, viruses,endotoxins, whole cells and cellular debris.

The terms “target molecule,” “target biomolecule,” “desired targetmolecule” and “desired target biomolecule,” as used interchangeableherein, generally refer to a polypeptide or product of interest, whichis desired to be purified or separated from one or more undesirableentities, e.g., one or more impurities, which may be present in a samplecontaining the polypeptide or product of interest. The terms “protein ofinterest,” “target polypeptide,” “polypeptide of interest,” and “targetprotein,” as used interchangeably herein, generally refer to atherapeutic protein or polypeptide, including but not limited to, anantibody that is to be purified using a stimulus responsive polymeraccording to the present invention.

As used herein interchangeably, the term “polypeptide” or “protein,”generally refers to peptides and proteins having more than about tenamino acids. In some embodiments, a stimulus responsive polymerdescribed herein is used to separate a protein or polypeptide from oneor more undesirable entities present in a sample along with the proteinor polypeptide. In some embodiments, the one or more entities are one ormore impurities which may be present in a sample along with the proteinor polypeptide being purified. As discussed, above, in some embodiments,a stimulus responsive polymer described herein specifically binds andprecipitates a protein or polypeptide of interest upon the addition of astimulus to the sample. In other embodiments, a stimulus responsivepolymer described herein binds to and precipitates an entity other thanthe protein or polypeptide of interest such as, for example, host cellproteins, DNA, viruses, whole cells, cellular debris and cell cultureadditives, upon the addition of a stimulus.

In some embodiments, a protein or polypeptide being purified using astimulus responsive polymer described herein is a mammalian protein,e.g., a therapeutic protein or a protein which may be used in therapy.Exemplary proteins include, but are not limited to, for example, renin;a growth hormone, including human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factors such as factor VIIIC,factor IX, tissue factor, and von Willebrands factor; anti-clottingfactors such as Protein C; atrial natriuretic factor; lung surfactant; aplasminogen activator, such as urokinase or human urine or tissue-typeplasminogen activator (t-PA); bombesin; thrombin; hemopoietic growthfactor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); a serum albumin such ashuman serum albumin; Muellerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; Dnase; IgE; a cytotoxicT-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;activin; vascular endothelial growth factor (VEGF); receptors forhormones or growth factors; Protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor.(BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-β; platelet-derived growth factor (PDGF);fibroblast growth factor such as α-FGF and β-FGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGFβ4, or TGF-β5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CDproteins such as CD3, CD4, CD8, CD19 CD20, CD34, and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (Ils), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as for example, a portion of theAIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments and/or variants of any of the above-listedpolypeptides.

Further, in some embodiments, a protein or polypeptide purified using asmart polymer according to the present invention is an antibody,functional fragment or variant thereof. In some embodiments, a proteinof interest is a recombinant protein containing an Fc region of animmunoglobulin.

The term “immunoglobulin,” “Ig or “antibody” (used interchangeablyherein) refers to a protein having a basic four-polypeptide chainstructure consisting of two heavy and two light chains, said chainsbeing stabilized, for example, by interchain disulfide bonds, which hasthe ability to specifically bind antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind antigen. The term “domain” refers to a globular region of a heavyor light chain polypeptide comprising peptide loops (e.g., comprising 3to 4 peptide loops) stabilized, for example, by β-pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of antibodylight chains are referred to interchangeably as “light chain constantregions”, “light chain constant domains”, “CL” regions or “CL” domains.The “constant” domains of antibody heavy chains are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains. The “variable” domains ofantibody light chains are referred to interchangeably as “light chainvariable regions”, “light chain variable domains”, “VL” regions or “VL”domains. The “variable” domains of antibody heavy chains are referred tointerchangeably as “heavy chain variable regions”, “heavy chain variabledomains”, “VH” regions or “VH” domains.

Immunoglobulins or antibodies may be monoclonal or polyclonal and mayexist in monomeric or polymeric form, for example, IgM antibodies whichexist in pentameric form and/or IgA antibodies which exist in monomeric,dimeric or multimeric form. Immunoglobulins or antibodies may alsoinclude multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they retain, or are modified to comprise,a ligand-specific binding domain. The term “fragment” refers to a partor portion of an antibody or antibody chain comprising fewer amino acidresidues than an intact or complete antibody or antibody chain.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. When produced recombinantly, fragmentsmay be expressed alone or as part of a larger protein called a fusionprotein. Exemplary fragments include Fab, Fab′, F(ab′)2, Fc and/or Fvfragments. Exemplary fusion proteins include Fc fusion proteins.

Generally, an immunoglobulin or antibody is directed against an“antigen” of interest. Preferably, the antigen is a biologicallyimportant polypeptide and administration of the antibody to a mammalsuffering from a disease or disorder can result in a therapeutic benefitin that mammal. However, antibodies directed against nonpolypeptideantigens (such as tumor-associated glycolipid antigens; see U.S. Pat.No. 5,091,178) are also contemplated. Where the antigen is apolypeptide, it may be a transmembrane molecule (e.g. receptor) or aligand such as a growth factor.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies arc highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). “Monoclonal antibodies” may also be isolatedfrom phage antibody libraries using the techniques described in Clarksonet al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example.

Monoclonal antibodies may further include “chimeric” antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sc. USA 81:6851-6855 (1984)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (LI),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications arc made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

In some embodiments, an antibody which is separated or purified using astimulus responsive polymer according to the present invention is atherapeutic antibody. Exemplary therapeutic antibodies include, forexample, trastuzumab (HERCEPTIN™, Genentech, Inc., Carter et al (1992)Proc. Natl. Acad. Sci. USA, 89:4285-4289; U.S. Pat. No. 5,725,856);anti-CD20 antibodies such as chimeric anti-CD20 “C2B8” U.S. Pat. No.5,736,137); rituximab (RITUXAN™), ocrelizumab, a chimeric or humanizedvariant of the 21-17 antibody (U.S. Pat. No. 5,721,108; WO 04/056312) ortositumomab (BEXXAR™); anti-IL-8 (St John et at (1993) Chest. 103:932,and WO 95/23865); anti-VEGF antibodies including humanized and/oraffinity matured anti-VEGF antibodies such as the humanized anti-VEGFantibody huA4.6.1 bevacizumab (AVASTAN™, Genentech, Inc., Kim et al(1992) Growth Factors 7:53-64, WO 96/30046, WO 98/45331); anti-PSCAantibodies (WO 01/40309); anti-CD40 antibodies, including S2C6 andhumanized variants thereof (WO 00/75348); anti-CDI la (U.S. Pat. No.5,622,700; WO 98/23761; Steppe et al (1991) Transplant. Intl. 4:3-7;Hourmant et al (1994) Transplantation 58:377-380); anti-IgE (Presta etal (1993) J. Immunol. 151:2623-2632; WO 95/19181); anti-CD18 (U.S. Pat.No. 5,622,700; WO 97/26912); anti-IgE, including E25, E26 and E27 (U.S.Pat. No. 5,714,338; U.S. Pat. No. 5,091,313; WO 93/04173; U.S. Pat. No.5,714,338); anti-Apo-2 receptor antibody (WO 98/51793); anti-TNF-alphaantibodies including cA2 (REMICADE™), CDP571 and MAK-195 (U.S. Pat. No.5,672,347; Lorenz et al (1996) J. Immunol. 156(4):1646-1653; Dhainaut etal (1995) Crit. Care Med. 23(9):1461-1469); anti-Tissue Factor (TF) (EP0 420 937 B1); anti-human alpha 4 beta 7 integrin (WO 98/06248);anti-EGFR, chimerized or humanized 225 antibody (WO 96/40210); anti-CD3antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or anti-tacantibodies such as CHI-621 SIMULECT™ and ZENAPAX™ (U.S. Pat. No.5,693,762); anti-CD4 antibodies such as the cM-7412 antibody (Choy et at(1996) Arthritis Rheum 39(1):52-56); anti-CD52 antibodies such asCAMPATH-1H (Riechmann et al (1988) Nature 332:323-337); anti-Fc receptorantibodies such as the M22 antibody directed against Fe gamma RI as inGraziano et al (1995) J. Immunol. 155(10):4996-5002;anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkeyet al (1995) Cancer Res. 55(23Suppl): 5935s-5945s; antibodies directedagainst breast epithelial cells including huBrE-3, hu-Mc 3 and CRL6(Ceriani et al (1995) Cancer Res. 55(23):5852s-5856s; and Richman et al(1995) Cancer Res. 55(23 Supp): 5916s-5920s); antibodies that bind tocolon carcinoma cells such as C242 (Litton et al (1996) Eur J. Immunol.26(1):1-9); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al (1995) J.Immunol. 155(2):925-937); anti-CD33 antibodies such as Hu M195 (Jurcicet al (1995) Cancer Res 55(23 Suppl):5908s-5910s and CMA-676 or CDP771:anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al (1995)Cancer Res 55(23 Suppl):5899s-5907s); anti-EpCAM antibodies such as17-1A (PANOREX™); anti-GpIIb/IIIa antibodies such as abciximab or c7E3Fab (REOPRO™); anti-RSV antibodies such as MEDI-493 (SYNAGIS™); anti-CMVantibodies such as PROTOVIR™); anti-HIV antibodies such as PRO542;anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR™);anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2;anti-alpha v beta3 antibody VITAXIN™; anti-human renal cell carcinomaantibody such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94);anti-human colorectal tumor antibody (A33); anti-human melanoma antibodyR24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma(SF-25); and anti-human leukocyte antigen (HLA) antibodies such as SmartID10 and the anti-HLA DR antibody Oncolym (Lym-1).

The terms “contaminant,” “impurity,” and “debris,” as usedinterchangeably herein, refer to any foreign or objectionable material,including a biological macromolecule such as a DNA, an RNA, one or morehost cell proteins (HCPs or CHOPs), endotoxins, viruses, lipids and oneor more additives which may be present in a sample containing a proteinor polypeptide of interest (e.g., an antibody) being separated from oneor more of the foreign or objectionable molecules using a stimulusresponsive polymer according to the present invention. In someembodiments, a stimulus responsive polymer described herein binds andprecipitates a protein or polypeptide of interest from a samplecontaining the protein or polypeptide of interest and one or moreimpurities. In other embodiments, a stimulus responsive polymerdescribed herein binds and precipitates one or more impurities, therebyto separate the polypeptide or protein of interest from one or moreimpurities.

The terms “chinese hamster ovary cell protein” and “CHOP,” as usedinterchangeably herein, refer to a mixture of host cell proteins (“HCP”)derived from a Chinese hamster ovary (“CHO”) cell culture. The HCP orCHOP is generally present as an impurity in a cell culture medium orlysate (e.g., a harvested cell culture fluid containing a protein orpolypeptide of interest (e.g., an antibody or immunoadhesin expressed ina CHO cell). Generally, the amount of CHOP present in a mixturecomprising a protein of interest provides a measure of the degree ofpurity for the protein of interest. Typically, the amount of CHOP in aprotein mixture is expressed in parts per million relative to the amountof the protein of interest in the mixture.

It is understood that where the host cell is another mammalian celltype, an E. coli, a yeast cell, an insect cell, or a plant cell, HCPrefers to the proteins, other than target protein, found in a lysate ofthe host cell.

The term “cell culture additive,” as used herein, refers to a molecule(e.g., a non-protein additive), which is added to a cell culture processin order to facilitate or improve the cell culture or fermentationprocess. In some embodiments according to the present invention, astimulus responsive polymer, as described herein, binds and precipitatesone or more cell culture additives. Exemplary cell culture additivesinclude anti-foam agents, antibiotics, dyes and nutrients.

The term “parts per million” or “ppm,” as used interchangeably herein,refers to a measure of purity of a desired target molecule (e.g., atarget protein or antibody) purified using a stimulus responsive polymerdescribed herein. Accordingly, this measure can be used either to gaugethe amount of a target molecule present after the purification processor to gauge the amount of an undesired entity. In some embodiments, theunits “ppm” are used herein to refer to the amount of an impurity in asolution, e.g., HCP or CHOP, in nanograms/milliliter of protein ofinterest in milligrams/milliliter (i.e., CHOP ppm=(CHOP ng/ml)/(proteinof interest mg/ml). When the proteins are dried (e.g., bylyophilization), ppm refers to (CHOP ng)/(protein of interest mg)).

The terms “isolating,” “purifying” and “separating,” are usedinterchangeably herein, in the context of purifying a target molecule(e.g., a polypeptide or protein of interest) from a composition orsample comprising the target molecule and one or more impurities, usinga stimulus responsive polymer described herein. In some embodiments, thedegree of purity of the target molecule in a sample is increased byremoving (completely or partially) one or more impurities from thesample by using a stimulus responsive polymer, as described herein,. Inanother embodiment, the degree of purity of the target molecule in asample is increased by precipitating the target molecule away from oneor more impurities in the sample.

In some embodiments, a purification process additionally employs one ormore “chromatography steps.” Typically, these steps may be carried out,if necessary, after the separation of a target molecule from one or moreundesired entities using a stimulus responsive polymer according to thepresent invention.

In some embodiments, a “purification step” to isolate, separate orpurify a polypeptide or protein of interest using a stimulus responsivepolymer described herein, may be part of an overall purification processresulting in a “homogeneous” or “pure” composition or sample, which termis used herein to refer to a composition or sample comprising less than100 ppm HCP in a composition comprising the protein of interest,alternatively less than 90 ppm, less than 80 ppm, less than 70 ppm, lessthan 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, lessthan 20 ppm, less than 10 ppm, less than 5 ppm, or less than 3 ppm ofHCP.

The term “clarification step,” as used herein, generally refers to oneor more initial steps in the purification of biomolecules. Theclarification step generally comprises removal of cells and/or cellulardebris using one or more steps including any of the following alone orvarious combinations thereof, e.g., centrifugation and depth filtration,precipitation, flocculation and settling. Clarification step generallyinvolves the removal of one or more undesirable entities and istypically performed prior to a step involving capture of the desiredtarget molecule. Another key aspect of clarification is the removal ofsoluble and insoluble components in a sample which may later on resultin the fouling of a sterile filter in a purification process, therebymaking the overall purification process more economical. In someembodiments, the present invention provides an improvement over theconventional clarification steps commonly used in various purificationschemes, as demonstrated by reduced turbidity/impurities and higherthroughput of downstream filters, described in the Examples herein.

The term “chromatography,” as used herein, refers to any kind oftechnique which separates an analyte of interest (e.g., a targetmolecule) from other molecules present in a mixture. Usually, theanalyte of interest is separated from other molecules as a result ofdifferences in rates at which the individual molecules of the mixturemigrate through a stationary medium under the influence of a movingphase, or in bind and elute processes.

The term “chromatography resin” or “chromatography media” are usedinterchangeably herein and refer to any kind of phase (e.g., a solidphase) which separates an analyte of interest (e.g., a target molecule)from other molecules present in a mixture. Usually, the analyte ofinterest is separated from other molecules as a result of differences inrates at which the individual molecules of the mixture migrate through astationary solid phase under the influence of a moving phase, or in bindand elute processes. Examples of various types of chromatography mediainclude, for example, cation exchange resins, affinity resins, anionexchange resins, anion exchange membranes, hydrophobic interactionresins and ion exchange monoliths.

The term “capture step” as used herein, generally refers to a methodused for binding a target molecule with a stimulus responsive polymer ora chromatography resin, which results in a solid phase containing aprecipitate of the target molecule and the polymer or resin. Typically,the target molecule is subsequently recovered using an elution step,which removes the target molecule from the solid phase, therebyresulting in the separation of the target molecule from one or moreimpurities. In various embodiments, the capture step can be conductedusing a chromatography media, such as a resin, membrane or monolith, ora polymer, such as a stimulus responsive polymer, polyelectolyte orpolymer which binds the target molecule.

The term “salt,” as used herein, refers to a compound formed by theinteraction of an acid and a base. Various salts which may be used invarious buffers employed in the methods described herein include, butare not limited to, acetate (e.g. sodium acetate), citrate (e.g., sodiumcitrate), chloride (e.g., sodium chloride), sulphate (e.g., sodiumsulphate), or a potassium salt.

The term “multivalent salt” or “multivalent ion,” as usedinterchangeably herein, refers to a compound which contains more thanone charge or a charge containing group. In some embodiments, amultivalent salt is used as a stimulus which results in changing thesolubility of a polymer responsive to the salt stimulus, usuallyresulting in precipitating the polymer out of solution. Exemplarymultivalent salts which may be used include, for example, phosphate andsulfate. Also encompassed by the present invention are counter ionswhich may be used such as, for example, citrate. A multivalent salt,when used a stimulus as described herein, may be added as a stand alonereagent to a sample containing a biomolecule of interest along with astimulus responsive polymer. Alternatively, the salt may be attached toa substrate, e.g., a membrane. In a particular embodiment, a membrane ismodified with polyvinylphosphate, which is a multivalent salt containingpolymer coating. A multivalent ion used as a stimulus, as describedherein, is considered less detrimental to protein structure andstability, relative to other stimuli such as temperature and pH.

In some embodiments, multivalent salts are capable of interacting withone or more entities, thereby to form an associated species or complex.Accordingly, such salts may also be referred to as “complex formingsalts.” Non-limiting examples of complex forming salts and theirresultant complexes are multivalent cations such as Cu²⁺ and Ca²⁺ andtheir complexes with the carboxylic acid groups found inethylenediaminetetraacetate; multivalent anions such as phosphate (PO₄³⁻) and citrate and their complexes with primary amines found inpolyallylamine; and ion-associating salts such as perchlorate, dodecylsulfate and dodecyl benzene sulfonate and their complexes with primaryamines found in polyallylamine.

An “ion-associating salt” is a univalent (cation or anion), bulky andcharge-dispersed salt. In some embodiments, an ion-associating salt isused as a stimulus which results in changing the solubility of a polymerresponsive to the salt stimulus, thereby resulting in precipitating thepolymer out of solution. Exemplary ion-associating salts which may beused include, for example, perchlorate, dodecyl sulfate, dodecyl benzenesulfonate, tetraphenyl borate and hexanitrodiphenol amine.

The term “solvent,” as used herein, generally refers to a liquidsubstance capable of dissolving or dispersing one or more othersubstances to provide a solution. Solvents include aqueous and organicsolvents, where useful organic solvents include a non-polar solvent,ethanol, methanol, isopropanol, acetonitrile, hexylene glycol, propyleneglycol, and 2,2-thiodiglycol.

The term “pI” or “isoelectric point” of a polypeptide, as usedinterchangeably herein, refers to the pH at which the polypeptide'spositive charge balances its negative charge. pI can be calculated fromthe net charge of the amino acid residues or sialic acid residues ofattached carbohydrates of the polypeptide or can be determined byisoelectric focusing.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Preparation of Unclarified Non-Expressing CellCulture Fluid (CCF)

In a representative experiment, cells derived from a non-expressingChinese Hamster Ovary (CHO) cell line were grown in a 10L bioreactor(New Brunswick Scientific) to a density of 10×10⁶ cells/mL and harvestedat 64% viability. IgG was spiked to a concentration of 1.3 g/L. Thelevel of host cell proteins (HCP) was found to be 8300 ng/mL using anELISA (Cygnus #CM015). The pH of the unclarified cell culture was pH7.2.

Example 2 Synthesis of a Stimulus Responsive Polymer Comprising aPolyelectrolyte Backbone Modified With a Hydrophobic Group

In a representative experiment, a stimulus responsive polymer comprisinga polyallylaminc (BzMPAA) backbone modified with a hydrophobic group wassynthesized. A polymer comprising a cationic polyelectrolyte backbonemodified with a hydrophobic group was synthesized using a mixture of10.3 g of 40% wt linear polyallylamine (NITTOBO, 150 kD), 2 g of lithiumhydroxide and 20 mL of 50% water/methanol, which was stirred until wellmixed. A solution of 2.1 mL of benzyl chloride in 15 mL of methanol wasadded to the polymer solution. The resulting mixture was heated at 60°C. for 14 hours. The benzyl modified polyallylamine was precipitated atthe end of the reaction period as a result of thermodynamicincompatibility with the solvent. The precipitate was washed With 30 mLof acetone and redissolved in 400 mL of 1M acetic acid. The polymer wasfurther purified by precipitation using 50 mM sodium phosphate at pH 7.FIG. 1 depicts a schematic of the reaction of the polyallylaminepolyelectrolye polymer with a hydrophobic group, i.e., benzylchloride.

Example 3 Preparation of a Solution of Benzyl Modified Polyallyamine(BzMPAA)

A 10% solution of BzMPAA was prepared by dissolving 10 g of the polymerfrom Example 2 in 90 g of 1M acetic acid with continuous agitation atroom temperature for 16 hours. The resulting viscous solution wasslightly hazy.

Example 4 Use of Different Concentrations of Benzyl ModifiedPolyallyamine (BxMPAA) in Clarification of a Non-Expressing Cell CultureFluid (CCF)

BzMPAA from Example 3 was added in amounts of 0.2 g, 0.3 g, 0.4 g and0.5 g to a 5 mL sample of the unclarified cell culture fluid (CCF) fromExample 1. The samples were mixed at room temperature for 2 minutes.Since polymer addition reduced the pFI to a range of pH 4.5 to 5.5, thepH of the mixtures were adjusted using 2M Tris base to a pH of 7. To theresulting solution, 0.043 g of potassium phosphate dibasic was added inorder to precipitate the polymer-target molecule, cell and cell debriscomplex. The precipitate, in the form of a dispersed solid suspensionwas mixed continuously for five minutes. The precipitate was thencollected via centrifugation (4000 rpm for 1 minute). The supernatantfrom each sample was then filtered through a 0.2 um Durapore® filter.The resulting purification is detailed in Table 1 below.

Example 5 Use of Different pH Values in the Clarification ofNon-Expressing CCF With BzMPAA

BzMPAA from Example 3 was added to four samples (0.4 g each) containing5 mL of the unclarified cell culture fluid from Example 1. The sampleswere mixed at room temperature for 2 minutes. Since polymer additionreduced the pH to a range of pH 4.5 to 5.5, the pH of the mixtures wereadjusted using 2M Tris base to pHs of 5.5, 6.5 7.5 and 8.5,respectively. To the resulting solution, 0.043 g of potassium phosphatedibasic was added in order to precipitate the polymer-target molecule,cell and cell debris complex. The precipitate, which was in the form ofa dispersed solid suspension, was mixed continuously for five minutes.The precipitate was then collected via centrifugation (4000 rpm for 1minute). The supernatant from each sample was then filtered through a0.2 um Durapore® filter. The resulting purification is detailed in Table1 below, which describes BzMPAA purification of spiked non-expressingCHO CCF.

TABLE 1 Polymer Concentration IgG Recovery HCP Removal DNA Removal (wt%) Ph (%) (%) (LRV) 0.4 7 97 63 2.7 0.6 7 92 82 2.8 0.8 7 86 83 2.7 1.07 82 85 2.7 0.8 5.5 95 83 3.0 0.8 6.5 86 91 2.9 0.8 7.5 90 86 2.8 0.88.5 95 62 2.9

Example 6 Assaying for the Purity Levels Resulting From the Use ofBzMPAA in the Cclarification of CCF

Samples from Example 4 and Example 5 were assayed for IgG recovery usingan affinity Protein A analytical HPLC assay. The level of IgG insolution was alternatively measured using an analytical Protein Acolumn. Specifically, a Poros A/20 Protein A column (Applied Biosystems)was equilibrated with PBS, eluted with 0.1 M lysine (pH 2) and cleanedwith 6M guanidine HCl. An IgG standard curve was created using a seriesof varying injection volumes of polyclonal IgG (Seracare). Samples wereinjected and IgG concentrations determined from the standard curve.

Samples from Example 4 and Example 5 were assayed for host cell proteins(HCP) using a commercial enzyme-linked immunosorbent assay (ELISA) kit(Cygnus Technologies Inc., Southport, N.C., Cygnus #CM015). Samples fromExample 4 and Example 5 were also assayed for DNA using a standard picogreen assay and Herring sperm DNA as a standard.

Example 7 Preparation of Unclarified Cell Culture Fluid (CCF)

Cells derived from an expressing Chinese Hamster Ovary (CHO-DG44) cellline were grown in a 10L bioreactor (New Brunswick Scientific) to adensity of 10×10⁶cells/mL and harvested at 30% viability. Monoclonalantibody (MAb) titer was determined to be 0.8 g/L. The level of hostcell proteins (HCP) was found to be 200,000 ng/mL using an ELISA assay(Cygnus #3G ELISA). The pH of the unclarified cell culture was pH 6.9.

Example 8 Synthesis of 20% Benzyl Modified Polyallylamine (BzMPAA)

10 g of polyallylamine (PAA) (Nittobo, 150 kD; 40% wt./wt.) was placedin a 100 mL round bottom flask and a solution of 3.34 g of sodiumhydroxide (1.2 Eq. per monomer) in 25 mL H₂O was added in small amountsat room temperature under magnetic stirring. The benzyl chloride (1.38g, 1.25 mL) was then added at once, stirred for few minutes at roomtemperature then heated to 60° C. overnight for 17 hours. The reactionwas cooled to room temperature and solvent was removed resulting inpolymer precipitation. The precipitated polymer was washed with waterand subsequently stirred in 1M aqueous AcOH solution (40 mL) untilcomplete solubilization is achieved. The solution was then diluted withH₂O to a final volume of 400 mL (1% polymer solution), potassium dibasicphosphate (K₂HPO4) (3.48 g) was added under stirring and pH of thesolution was adjusted to about 6.8 to precipitate the modified polymer.The polymer was collected by filtration over a fritted funnel andfinally dried overnight in a vacuum oven overnight at 50-60° C.

Example 9 Synthesis of 40% Benzyl Modified Polyallylamine (BzMPAA)

10 g of polyallylamine (PAA) (Nittobo, 150 kD; 40% wt./wt.) was placedin a 100 mL round bottom flask and a solution of 3.34 g of sodiumhydroxide (1.2 Eq. per monomer) in 25 mL H₂O was added at roomtemperature under magnetic stirring and in small amounts. Benzylchloride (2.30 g, 2.09 mL) was then added, stirred for few minutes atroom temperature and then heated to 60° C. overnight for 17 hours. Thereaction was then cooled to room temperature and solvent is removedresulting in polymer precipitation. The precipitated polymer was washedwith water and stirred in 1M aqueous AcOH solution (40 mL) untilcomplete solubilization is achieved. The solution was then diluted withH₂O to a final volume of 400 mL (1% polymer solution), potassium dibasicphosphate (K₂HPO4) (3.48 g) is added under stirring and pH of thesolution was adjusted to pH 6.8 to precipitate the modified polymer. Thepolymer was collected by filtration over a fritted funnel and finallydried overnight in a vacuum oven overnight at 50-60° C.

Example 10 Synthesis of 60% benzyl modified polyallylamine (BzMPAA)

10 g of polyallylamine (PAA) (NITTOBO, 150 kD; 40% wt./wt.) was placedin a 100 mL round bottom flask and a solution of 3.34 g of sodiumhydroxide (1.2 Eq. per monomer) in 25 mL H₂O was added at roomtemperature under magnetic stirring and in small amounts. Benzylchloride (3.23 g, 2.94 mL) was then added and stirred for few minutes atroom temperature and subsequently heated to 60° C. overnight for 17hours. The reaction was then cooled to room temperature and solvent wasremoved. The precipitated polymer was washed with water then stirred in1M aqueous AcOH solution (40 mL) until complete solubilization isachieved. The solution was then diluted with H₂O to a final volume of400 mL (1% polymer solution), potassium dibasic phosphate (K₂HPO4) (3.48g) is added under stirring and pH of the solution was adjusted to pH 6.8to precipitate the modified polymer. The polymer was collected byfiltration over a fritted funnel and finally dried overnight in a vacuumoven overnight at 50-60° C.

Example 11 Synthesis of Diphenyl Modified Polyallylamine (DPhMPAA)

In an exemplary experiment, a polyelectrolyte polymer backbone,polyallylamine, was modified with a diphenyl group. Briefly, 10 g ofpolyallylamine (PAA) (Nittobo, 150 kD; 40% wt./wt.) was placed in a 100mL round bottom flask and a solution of 3.34 g of sodium hydroxide (1.2Eq. per monomer) in 25 mL H₂O is added at room temperature undermagnetic stirring in small amounts. The chloro-diphenyl methane (3.68 g,3.23 mL) was subsequently added, stirred for few minutes at roomtemperature then heated to 60° C. overnight for 17 hours. The reactionwas subsequently allowed to cool to room temperature and the solvent wasremoved. The precipitated polymer was washed with water and stirred in1M aqueous AcOH solution (40 mL). The remaining white solid, generatedby hydrolysis of the diphenyl chloromethane, was filtered out. Thesolution was subsequently diluted with H₂O to a final volume of 400 mL(1% polymer solution), potassium dibasic phosphate (K₂HPO4) (3.48 g) wasadded under stirring and pH of the solution is adjusted to pH 6.8 toprecipitate the modified polymer. The polymer was collected byfiltration over a fritted funnel and finally dried overnight in a vacuumoven overnight at 50-60° C.

Example 12 Synthesis of 6% Dichlorobenzyl Modified Polyallylamine(DClBzMPAA)

In another experiment, 10 g of polyallylamine (PAA) (NITTOBO, 150 kD;40% wt./wt.), was placed in a 100 mL round bottom flask and a solutionof 3.34 g of sodium hydroxide (1.2 Eq. per monomer) in 25 mL of H₂O isadded at room temperature under magnetic stirring and in small amounts.3,4-Dichlorobenzyl chloride (1.71 g, 1.21 mL) was subsequently added andthe mixture stirred at room temperature overnight for 17 hours. Thereaction mixture was subsequently diluted with 100 ml of H₂O, afterwhich pH is adjusted to neutral (pH 7.0) with phosphoric acid. Theprecipitated polymer was filtered out, washed with H₂ O and dried in avacuum oven overnight at 60° C. The polymer was collected by filtrationover a fritted funnel and finally dried overnight in a vacuum ovenovernight at 50-60° C.

Example 13 Synthesis of 10% Dichlorobenzyl Modified Polyallylamine(DCIBzMPAA)

In another representative experiment, 10% dichlorobenzyl modifiedpolyallylamine was synthesized as follows. 5 g polyallylamine (PAA)(NITTOBO, 150 kD; 40% wt./wt.) was placed in a 100 mL round bottom flaskand a solution of 1.68 g of sodium hydroxide (1.2 Eq. per monomer) in 40mL 50/50 H₂O/1,2 dimethoxyethane (DME) was added at room temperatureunder magnetic stirring and in small amounts. 3,4-dichlorobenzylchloride (0.57 g, 0.40 mL) was subsequently added, stirred for fewminutes at room temperature and then heated to 60° C. overnight for 21hours. The reaction was subsequently allowed to cool to roomtemperature, DME was removed under vacuum at 60-70° C. and then theremaining solvent is removed. The precipitated polymer was washed withwater and then stirred in 1M aqueous AcOH solution (20 mL) untilcomplete solubilization is achieved. The solution was then diluted withH₂O to a final volume of 200 mL (1% polymer solution), potassium dibasicphosphate (K₂HPO4) (1.74 g) was added under stirring and pH of thesolution was adjusted to pH 6.8 to precipitate the modified polymer. Thepolymer was collected by filtration over a fritted funnel and finallydried overnight in a vacuum oven at 50-60° C.

Example 14 Synthesis of 33% Chlorobenzyl Modified Polyallylamine(DCIBzMPAA)

In another representative experiment, a 33% chlorobenzyl modifiedpolyallylamine was synthesized as follows. 5 g polyallylamine (PAA)(NITTOBO, 150 kD, 40% wt./wt.) was placed in a 100 mL round bottom flaskand a solution of 3.34 g of sodium hydroxide (1.2 Eq. per monomer) in 40mL 50/50 H₂O/1,2 dimethoxyethane (DME) was added at room temperatureunder magnetic stirring and in small amounts. 4-chlorobenzyl chloride(1.48 g) was subsequently added, stirred for few minutes at roomtemperature and then heated to 60° C. overnight for 21 hours. The nextday, DME was evaporated under vacuum at 60-70° C. and the remainingsolvent was separated from the precipitated polymer. This latter waswashed with water then stirred in 1M aqueous AcOH solution (20 mL) untilcomplete solubilization is achieved. The solution was subsequentlydiluted with H₂O to a final volume of 200 mL (1% polymer solution),potassium dibasic phosphate (K₂HPO₄) (1.74 g) was added under stirringand pH of the solution is adjusted to pH 7 to precipitate the purifiedpolymer. The polymer was collected by filtration over a fritted funneland finally dried overnight in a vacuum at 50-60° C.

Example 15 Synthesis of 13% Phenyl-Benzyl Modified Polyallylamine(DCIBzMPAA)

In another experiment 13% phenyl-benzyl modified polyallylamine wassynthesized as follows. 4.7 g of polyallylamine (PAA) (Nittobo, 150 kD;40% wt./wt.) was placed in a 100 mL round bottom flask and a solution of3.34 g of sodium hydroxide (1.2 Eq. per monomer) in 40 mL 50/50 H₂O/1,2dimethoxyethane (DME) was added at room temperature under magneticstirring and in small amounts. 4-phenylbenzyl chloride (1 g) was thenadded and the mixture heated overnight at 55° C. for 20 hours. Thereaction was subsequently allowed to cool to room temperature, DME wasremoved under vacuum at 60-70° C. and the remaining solvent was removedfrom the precipitated polymer. This latter was washed with water thenstirred in 1M aqueous AcOH solution (40 mL) overnight to complete thesolubilization. The solution was subsequently diluted with 1-170 to afinal volume of 200 mL (1% polymer solution), potassium dibasicphosphate (K₂HPO₄) (1.74 g) was added under stirring and pH of thesolution was adjusted to pH 7.0 in order to precipitate the purifiedpolymer. The polymer was collected by filtration over a fritted funneland finally dried overnight in a vacuum at 50-60° C.

Example 16 Synthesis of 27% Phenyl-Benzal Modified Polyallylamine(DClBzMPAA)

In another experiment, 27% phenyl-benzyl modified polyallylamine wassynthesized as follows. 2.8 g polyallylamine (PAA) (Nittobo, 150 kD; 40%wt./wt.) was placed in a 100 mL round bottom flask and a solution of3.34 g of sodium hydroxide (1.2 Eq. per monomer) in 24 mL 50/50 H₂O/1,2dimethoxyethane (DME) was added at room temperature under magneticstirring and in small amounts. 4-phenylbenzyl chloride (1 g) wassubsequently added and the mixture was heated at 55° C. overnight for 20hours. The reaction was subsequently allowed to cool to roomtemperature, DME was removed under vacuum at 60-70° C. and the remainingsolvent was removed from the precipitated polymer. This latter waswashed with water then stirred in 1M aqueous AcOH solution (32 mL) andstirred overnight to complete the solubilization. The solution wassubsequently diluted with H₂O to a final volume of 200 mL (1% polymersolution), potassium dibasic phosphate (K₂HPO₄) (1.74 g) was added understirring and pH of the solution was adjusted to pH 7.0 to precipitatethe purified polymer. The polymer was collected by filtration over afritted funnel and finally dried overnight in a vacuum at 50-60° C.

Example 17 Clarification of CCF with Different Polymer Concentrations

In an exemplary experiment, stimulus responsive polymers, describedabove in Examples 8-16, were evaluated for clarification of CCF,preparation of which is described above in Example 7. A polymer solutionfrom Examples 8-16 was added in amounts of 0.2 g, 0.3 g, 0.4 g and 0.5 gto a 5 mL sample of the unclarified cell culture fluid. The samples weremixed at room temperature for 2 minutes. Since polymer addition reducedthe pH to a range of pH 4.5 to 5.5, the pH of the mixtures Were adjustedusing 2M Tris base to a pH of 7. To the resulting solution, 0.043 g ofpotassium phosphate dibasic was added in order to precipitate thepolymer-target molecule, cell and cell debris complex. The precipitate,in the form of a dispersed solid suspension, was mixed continuously forfive minutes. The precipitate was then collected via centrifugation(4000 rpm for 1 minute). The supernatant from each sample wassubsequently filtered through a 0.2 μm Durapore® filter. The resultingpurification is detailed in Table 2.

Example 18 Clarification of CCF with Polymers at Different pH

In another experiment, a polymer solution from Examples 8-16 was addedto four samples (0.4 g each) containing 5 mL of the unclarified cellculture fluid, as described in Example 7. The samples were mixed at roomtemperature for 2 minutes. Since polymer addition reduced the pH to arange of pH 4.5 to 5.5, the pH of the mixtures were adjusted using 2MTris base to pHs of 5.5, 6.5 7.5 and 8.5, respectively. To the resultingsolution, 0.043 g of potassium phosphate dibasic was added in order toprecipitate the polymer-target molecule, cell and cell debris complex.The precipitate, in the form of a dispersed solid suspension, was mixedcontinuously for five minutes. The precipitate was then collected viacentrifugation (4000 rpm for 1 minute). The supernatant from each samplewas subsequently filtered through a 0.2 μm Durapore® filter. Theresulting purification is described in Table 2.

Example 19 Clarification of CCF Using Polymers Responsive to DifferentLevels of Multivalent ion Stimulus

In another experiment, polymers described in Examples 8-15 wereevaluated for the clarification of CCF, using different amounts ofmultivalent ion stimulus. Specifically, polymers from Examples 8-15 wereadded to four samples (0.4 g each) containing 5 mL of the unclarifiedcell culture fluid, described in Example 7. The samples were mixed atroom temperature for 2 minutes. Since polymer addition reduced the pH toa range of pH 4.5 to 5.5, the pH of the mixtures were adjusted using 2MTris base to pHs of 5.5, 6.5 7.5 and 8.5, respectively. To the resultingsolution, a range from 0.031 to 0.043 g (50 to 70 mM final phosphateconcentration) of potassium phosphate dibasic was added in order toprecipitate the polymer-target molecule, cell and cell debris complexes.The precipitate, in the form of a dispersed solid suspension, was mixedcontinuously for five minutes. The precipitate was subsequentlycollected via centrifugation (4000 rpm for 1 minute). The supernatantfrom each sample was then filtered through a 0.2 μm Durapore® filter.The resulting purification is described in Table 2.

Example 20 Clarification of CCF Using a Commonly Used Flocculant

In another experiment, a commonly used flocculant, chitosan, was usedfor the clarification of CCF, in an attempt to generate comparativedata. A polymer solution (2 wt %) was made according to the proceduredescribe in Riske, F. et.al.; Journal of Biotechnology, 128 (2007)813-823. The polymer solution was added to CCF from Example 7 in varyingamounts and at varying pH conditions. No stimulus was used with thispolymer. The resulting purification is described in Table 2.

Example 21 Evaluation of Purity Levels Following Precipitation of CCFUsing Stimulus Responsive Polymers

In a representative experiment, polymers described in Examples 8-16 wereassayed for IgG recovery using an affinity Protein A analytical HPLCassay. The level of IgG in solution was alternatively measured using ananalytical Protein A column. A Poros A/20 Protein A column (AppliedBiosystems) was equilibrated with PBS, eluted with 0.1 M lysine (pH 2)and cleaned with 6M guanidine HCl. An IgG standard curve was createdusing a series of varying injection volumes of polyclonal IgG(Seracare). Samples were injected and IgG concentrations determined fromthe standard curve. Samples from Examples 8-16 were assayed host cellproteins (HCP) using a commercial enzyme-linked immunosorbent assay(ELISA) kit (Cygnus Technologies Inc., Southport, N.C., Cygnus #3G).Samples from Examples 8-16 were assayed for DNA using a standard picogreen assay and Herring sperm DNA as a standard. To assess the reductionof cells and cell debris, the turbidity was measured aftercentrifugation for 1 minute at 4000 rpm.

TABLE 2 Characterization Data for Examples 8-16 and 20. PolymerMultivalent IgG HCP DNA Conc. Salt Conc. Turbidity Recovery RemovalRemoval Example (wt %) (mM) pH (NTU) (%) (%) (%) 8 0.4 70 7.5 26.7 88 5895 8 0.6 70 7.5 12.8 86 65 95 8 0.8 62 7.5 31.4 79 72 97 8 1.0 62 7.5104 79 75 94 8 0.8 62 8.5 7.8 71 67 95 8 0.8 62 6.5 60.8 86 76 96 8 0.862 5.5 76.5 99 76 97 9 0.4 70 7.5 2.2 82 83 98 9 0.6 70 7.5 2.5 6485 >99 9 0.8 62 7.5 2.8 61 87 >99 9 1.0 62 7.5 2.8 53 88 >99 9 0.8 628.5 7.1 79 80 >99 9 0.8 62 6.5 3.0 58 91 98 9 0.8 62 5.5 3.4 87 92 97 100.4 70 7.5 5.1 54 88 95 10 0.6 70 7.5 4.9 26 90 98 10 0.8 62 7.5 5.6 2193 >99 10 1.0 62 7.5 1.9 18 93 >99 10 0.8 62 8.5 6.1 40 89 >99 10 0.8 626.5 5.0 14 96 >99 10 0.8 62 5.5 2.8 26 95 >99 11 0.4 70 7.5 124 97 42 9811 0.6 70 7.5 210 61 52 98 11 0.8 62 7.5 478 92 56 99 11 1.0 62 7.5 47484 55 98 11 0.8 62 8.5 94.2 54 65 98 11 0.8 62 6.5 380 88 60 97 11 0.862 5.5 212 99 58 97 12 0.6 70 7.5 3.4 50 74 >99 12 0.8 62 7.5 1.9 8558 >99 12 1.0 62 7.5 1.3 70 66 >99 12 0.8 62 8.5 1.4 33 81 >99 12 0.8 626.5 1.0 37 93 >99 12 0.8 62 5.5 1.4 93 77 >99 12 0.6 50 7.5 1.7 8185 >99 12 0.6 50 6.5 3.1 87 80 >99 12 0.4 50 5.5 1.6 98 66 >99 13 0.4 707.5 2.3 86 71 98 13 0.6 70 7.5 1.9 71 73 98 13 0.8 62 7.5 2.4 81 77 >9913 1.0 62 7.5 1.4 68 80 >99 13 0.8 62 8.5 1.5 60 69 >99 13 0.8 62 6.51.1 77 87 >99 13 0.8 62 5.5 1.3 98 90 >99 13 0.6 50 7.5 1.7 78 84 >99 130.6 50 6.5 3.1 39 84 >99 13 0.4 50 5.5 1.6 69 78 >99 14 0.4 70 7.5 5.469 91 >99 14 0.6 70 7.5 7.3 46 89 >99 14 0.8 62 7.5 5.1 57 94 >99 14 1.062 7.5 4.3 22 87 >99 14 0.8 62 8.5 2.8 41 92 >99 14 0.8 62 6.5 3.7 297 >99 14 0.8 62 5.5 8.5 7 94 >99 14 0.6 50 7.5 7.5 44 89 >99 14 0.6 506.5 6.5 57 96 >99 14 0.4 50 5.5 6.6 43 90 >99 15 0.4 70 7.5 3.0 9375 >99 15 0.6 70 7.5 1.5 ~100 86 >99 15 0.8 62 7.5 2.7 91 72 >99 15 1.062 7.5 2.4 67 91 >99 15 0.8 62 8.5 2.3 ~100 77 >99 15 0.8 62 6.5 1.4 5787 >99 15 0.8 62 5.5 2.0 89 90 >99 15 0.6 50 7.5 5.3 ~100 88 >99 15 0.650 6.5 6.3 ~100 70 >99 15 0.4 50 5.5 7.9 ~100 83 >99 16 0.4 70 7.5 9.485 77 >99 16 0.6 70 7.5 9.9 54 93 >99 16 0.8 62 7.5 9.7 ~100 85 >99 161.0 62 7.5 19.0 67 94 >99 16 0.8 62 8.5 9.1 ~100 84 >99 16 0.8 62 6.55.6 78 94 >99 16 0.8 62 5.5 4.2 54 88 >99 16 0.6 50 7.5 7.2 56 90 >99 160.6 50 6.5 4.1 89 92 >99 16 0.4 50 5.5 2.6 85 89 >99 20 1 0 7.5 27.0~100 39 30 20 0.8 0 8.5 22.0 61 63 54 20 0.8 0 6.5 16.0 70 30 34 20 0.80 5.5 195.0 63 62 96

Example 22 Synthesis of Benzyl Modified Polyethyleneimine (BzMPEI)

In another experiment, a benzyl modified polyethyleneimine stimulusresponsive polymer was synthesized as follows. 10 g PEI (Aldrich, 750kD; 50% wt./wt.) is placed in a 100 mL round bottom flask and a solutionof ˜3.34 g of sodium hydroxide (1.2 Eq. per monomer) in 25 mL H₂O isadded at room temperature under magnetic stirring and in small amounts.Benzyl chloride (2.30 g, 2.09 mL) is then added, stirred for few minutesat room temperature and then heated overnight at 60° C. for 17 hours.The reaction is then allowed to cool to room temperature and the solventis removed. The precipitated polymer is washed with water then stirredin 1M aqueous AcOH solution (40 mL) until complete solubilization isachieved. The solution is subsequently diluted with H₂O to a finalvolume of 400 mL (1% polymer solution), potassium dibasic phosphate(K₂HPO4) (3.48 g) is added under stirring and pH of the solution isadjusted to pH 6.8 to precipitate the modified polymer. The polymer iscollected by filtration over a fritted funnel and finally driedovernight in a vacuum oven at 50-60° C.

Example 23 Synthesis of Benzyl Modified Polyvinylamine (BzMPVA)

In a representative example, a benzyl modified polyvinylamine wassynthesized as follows. 32 g of Poly(vinylamine) (PVA) hydrochloride (MW83,500, Air Products and Chemicals Inc.) was weighed into a glasscontainer. Add 200 mL H₂O and add 26 g 50% NaOH and stir until wellmixed. Add:23.5 g benzyl chloride and mix at 70° C. for 16 hrs. A solidwhite mass separates from supernatant as the reaction progresses. Allowsolid to settle and discard supernatant by decanting. Dissolve solid in350 mL of 3% Acetic Acid overnight. Add 360 mL of H₂O and mix for 16 hrsuntil solution is homogenous. Dilute up to a 1% w/v solution by bringingthe total volume up to 3.2L with deionized (DI) H₂O. Add sodiumphosphate to a concentration of 50 mM in order to initiate precipitationof the polymer. Add 1M NaOH to reach a pH of 6.8, providing additionalpolymer precipitation. Filter to dry cake and discard supernatant. Drysolid overnight at 70° C. Dissolve in 3% acetic acid until a homogenoussolution is generated in order to make a 5% w/v solution.

Example 24 Comparison of Modified and Unmodified Polymer inClarification in Presence of Stimulus

Polyallyamine (PAA, Nittobo, 150 kD; 40% wt./wt.) and the polymer fromExample. 10 (Bz-MPAA) were added to an aqueous solution to create afinal polymer concentration of 0.2% wt and 0.4%, respectively. Potassiumhydrogen phosphate was used as a stimulus and was added to the polymersolutions of PAA and Example 10 in varying amounts and the turbidity andnature of the aggregates formed was recorded. The results are shown inTable 3 below.

TABLE 3 Stimulus (mM Potassium Hydrogen Turbidity Nature of PolymerPhosphate) (NTU) Aggregate PAA 2.4 2.1 Clear (No Agg) PAA 4.7 3 Clear(No Agg) PAA 9.35 17.9 Clear (No Agg) PAA 18.7 41.2 Turbid PAA 37.5 152Turbid PAA 75 764 Milky/Opaque PAA 150 760 Milky/Opaque Example 10 2.4 1Clear (No Agg) (BzMPAA) Example 10 4.7 2 Clear (No Agg) (BzMPAA) Example10 9.35 4 Clear (No Agg) (BzMPAA) Example 10 18.7 9 Clear (No Agg)(BzMPAA) Example 10 37.5 28 Clear (No Agg) (BzMPAA) Example 10 75 230Large Aggregates (BzMPAA) Example 10 150 231 Large Aggregates (BzMPAA)

Example 25 Comparison of Modified and Unmodified Polymer inClarification in Presence of a Stimulus

Unmodified polymers polyallyamine (PAA, Nittobo, 150 kD; 40% wt./wt.),polyethyleneimine (PEI, Aldrich, 750 kD; 50% wt./wt.), polyvinyl amine(Poly(vinylamine) (PVA) hydrochloride, MW 83,500, Air Products andChemicals Inc.) and the modified polymers from Examples 10 (Bz-MPAA),Example 22 (BzMPEI) and Example 23 (BzMPVA) were added to an aqueoussolution to create a final polymer concentration of 0.2% and 0.4% byweight, respectively. Potassium hydrogen phosphate (150 mM) was used asa stimulus and was added to each of 0.4% polymer solutions and theturbidity and nature of the aggregates formed was recorded. The resultsare shown in Table 4.

TABLE 4 Stimulus Turbidity Turbidity Nature of Turbidity (Potassiumbefore after Aggregate after Hydrogen Stimulus Stimulus after CentrifugePolymer Phosphate) (NTU) (NTU) Stimulus (NTU) PAA 150 mM 0.1 >1000 Milky348 PEI 150 mM 2.9 >1000 Milky >1000 PVA 150 mM 0.5 >1000 Milky 233Example 9 150 mM 1.9 >1000 Large 9.6 (BzMPAA) Aggregates Example 18 150mM 5.1 >1000 Milky with 385 (BzMPEI) Large Aggregates Example 19 150 mM3.2 >1000 Large 12.7 (BzMPVA) Aggregates

Example 26 Synthesis of Hexanoic Acid and Tert-Butyl ModifiedPolyallylamine (HC-t-BuMPAA)

3.49 g of 6-bromohexanoic acid was dissolved in a solution comprising 10ml of 40 wt % linear polymer poly(allylamine)(150 kda, NITTOBO) and 30ml sodium hydroxide (1M). The mixture was reacted for 18 hrs at T=50° C.and the product was precipitated as a hydrated gel. The precipitate wasdissolved in 100 mg/ml lithium hydroxide solution and mixed with 10 mlmethanol containing 2.5 ml of tert-butyl glycidyl ether. The mixture wassubsequently reacted for 18 hrs at T=50° C. The polymer solution waspurified by extensive dialysis (3 days) against deionized (DI) waterusing 3.5 kda molecular weight cutoff dialysis tubing. The finalconcentration of the polymer solution was 7.2 wt %. A schematic of thesynthesis reaction is shown below in FIG. 2.

Example 27 Effect of Sodium Chloride on the Multivalent Stimulus ofHexanoic Acid and Tert-Butyl Modified Polyallylamine (HC-t-BuMPAA) whenDissolved in Tris Buffer

600 μl of HC-t-BuMPAA from Example 26 was added to 10 ml of 25 mM Sodiumphosphate containing 0, 0.15 or 0.5 M sodium chloride. Final pH of thesolution was 11.6. The solution was titrated with 3M acetic acid, andthe turbidity of the solution was recorded after each addition. Asdepicted in FIG. 3, by adding sodium chloride in addition to sodiumphosphate, a change in pH responsiveness, at which the phase transitionoccurs, was observed.

Example 28 Clarification of CCF Using the Polymer HC-t-BuMPAA atDifferent Polymer Concentrations

In a representative experiment, unclarified CCF was clarified using apolymer according to the present invention as follows. 178, 356, or 534μl of HC-t-BuMPAA from Example 26 was added to 5 ml of un-clarified cellculture fluid from Example 1 containing 25 mM sodium phosphate andadjusted to pH 8.7 using 25 μl of 3Macetic acid. After the addition ofthe polymer, final pH of the solution was titrated to 7.2 using 3Macetic acid, thereby precipitating the polymer-cells complex.

The precipitate, in the form of a dispersed solid suspension, was mixedcontinuously for another 5 min. The precipitate was then collected bycentrifugation (4000 rpm for 1 min) and the supernatant was filteredthrough a 0.2 μm Durapore® filter. Regardless of the concentration ofthe polymer used, the process resulted in 100% Mab recovery.

Example 29 Synthesis of Polyvinylamine (PVA) Stimulus Responsive Polymerfrom Monomer

A stimulus responsive polymer comprised of repeat units containingprimary amines was synthesized from a monomer as follows. 165 g ofdeionized water and 22.5 g of N-vinylformamide (NVF) (SIGMA-ALDRICH,98%) was placed in a 250 mL round bottom flask. The flask was equippedwith a magnetic stirrer and a N₂ dipstick. The solution was stirred andpurged with N₂ for 0.5 hours followed by heating to 45° C. over anadditional 0.5 hours with continuous purging. Initiator solution wasprepared by adding 0.288 g of 2,2’Azobis(2-amidinopropane)dihydrochloride (ABAP) (Aldrich) to 10 mL ofdeionized water and dissolved. To the 250 mL round bottom flask theinitiator solution was added under N₂ atmosphere. The solution washeated at 55° C. for 1 hour, followed by heating at 65° C. for 2 hours,further followed by heating at 75° C. for 1, hour with vigorous stirringunder nitrogen. A viscous, homogenous solution was obtained and allowedto cool to room temperature. The viscosity was determined by BrookfieldViscosity DV-II+ Pro Visocometer (setting 100 RPM, 45% torque, spindle#34). The viscosity of the resulting solution was 278-350 centipoise(cP). The solution was transferred to a 500 mL flask and diluted with330 mL of H₂0 and 40 g of 50% NaOH was added with stirring. The solutionwas heated at 85° C. for 8 hours.

A small sample was tested for sensitivity to a phosphate stimulus byadding a drop of 2 molar sodium phosphate to the hydrolyzed polymer andobserving a white solid precipitate from the solution upon addition ofthe phosphate ion. To the hydrolyzed polymer solution, 25% HCl was addeddrop wise, until a pH of around 2 was reached. The solution was stirredvigorously overnight and a homogenous yellow solution was obtained. Tothat solution, 100 mL of 4 molar NaOH was added with stirring along with500 mL isopropyl alcohol. The polymer was isolated by addition of 100 mLof 2 molar sodium phosphate and the solid was vacuum filtered and washedwith deionized water. The solid polymer was dried over night in a vacuumoven at 65° C. The partially dry polymer was frozen with liquid nitrogenand ground to a fine powder and further dried in a vacuum oven at 65° C.for 24 hours. Finally, 40.7 g of a dry powder was recovered anddissolved in 1 molar acetic acid to a final concentration of 5% w/w.

An overview of the polyvinylamine synthesis process is depicted in FIG.4.

Example 30 Synthesis of a Series of Hydrophobically ModifiedPolyvinylamine (PVA) Stimuli Responsive Polymers

Using the PVA synthesized from Example 29, three separatehydrophobically modified stimuli responsive polymers were produced asfollows. 100 mL of the 5% PVA solution from Example 29 was placed ineach of three 500 mL glass jars and labeled jars 1, 2, and 3. To each ofthe three jars, 100 g of 4 molar NaOH was added with stirring. Next, 50g of 1-propanol was added to each jar as a co-solvent and the solutionswere stirred, followed by the addition of 0.74 g, 1.47 g, and 2.94 g ofbenzyl chloride to jars 1, 2, and 3, respectively. The three jars wereheated at 60° C. for 16 hours. The resulting polymers were each isolatedfrom the individual reaction solutions by bringing the volume of thereaction solution to 500 mL with deionized water, adjusting the pH to 8with 25% HCl, and adding 100 g of 2 molar sodium phosphate. Uponaddition of the phosphate ion, a solid precipitate was collected viavacuum filtration. The solid from each reaction was individually washedwith deionized water and dissolved into 300 mL of 1 molar acetic acid.The polymer was further purified by adjusting the individual solutionsto pH 7.4, precipitating the polymer with drop wise addition of 2 molarsodium phosphate, filtering the resulting solids, washing the solidswith water, followed isopropyl alcohol, and drying in a vacuum oven at65° C. for 2 days. Each sample of dried polymer was frozen with liquidnitrogen and ground to a fine powder. The mass of dried polymerrecovered was 1.44, 2.12, and 2.47 g for jars 1, 2, and 3, respectively.The individual polymers were each dissolved to make a 2% solution in 1molar acetic acid.

Example 31 Deprotection of Amines via Hydrolysis of a Very HighMolecular Weight Poly(N-vinylacetamide) to Produce a Polyvinylamine(PVA) Stimuli Responsive Polymer

In another exemplary experiment, a very high molecular weight stimulusresponsive polymer was prepared as follows. Very high molecular weightpolymer generally refers to a polymer having a molecular weight equal orgreater than 1000 KDa.

A stimuli responsive polymer comprised of repeat units containingprimary amines was prepared as follows. In a 2 liter glass jar 40 g ofpoly(N-vinylacetamide)-linear homopolymer (POLYSCIENCES, INC:) with anaverage molecular weight of 4,060 kDa was dissolved into 0.8 liter ofdeionized water by vigorous stirring for 16 hours. To this solution, 140g concentrated HCl was added with continuous stirring over 1 hour. Thejar was lightly capped and the solution was heated to 99° C. for 5 dayswith intermittent rotation to mix the solution. After 5 days of heating,the solution was allowed to cool to room temperature and the totalvolume was adjusted to 4 liters with deionized water. The solution wasadjusted to pH 7 with 8 molar NaOH with vigorous stirring. Thehydrolyzed product was precipitated with drop wise addition of 2 molarsodium phosphate until no further precipitate was observed. The whiteprecipitate was washed with deionized water and pressed to remove excesswater. The recovered polymer was dried in a vacuum oven at 65° C. for 2days. The dried polymer was frozen with liquid nitrogen and ground to afine powder. The recovered dry mass was 42.5 g. A 2% solution was madeby dissolving the dried powder into 1 molar acetic acid and 0.08% HCl.The resulting solution was compared to a 2% solution of startingmaterial (poly(N-vinylacetamide)-linear homopolymer) for response to aphosphate or citrate stimuli. This was performed by drop wise additionof 2 molar sodium phosphate or 0.2 molar sodium citrate to 50 ml samplesof both the starting material and the resulting hydrolyzed polymer.

The resulting hydrolyzed polymer precipitated to a white mass with theaddition of phosphate or citrate ions while the solution of startingmaterial had no precipitate upon drop wise addition of phosphate orcitrate ions, thereby indicating that the starting material is notresponsive to a stimulus (e.g., a multivalent anion such as phosphate orcitrate), while the very high molecular weight polymer synthesized, asdescribed in this Example, is responsive to a stimulus (e.g., amultivalent anion such as phosphate or citrate).

FIG. 5 depicts a process for deprotection of polyamine polymer, therebyresulting in the formation of a stimulus responsive Polyvinylamine(PVA).

Example 32 Synthesis of a Hydrophobically Modified Polyvinylamine (PVA)Stimuli Responsive Polymer Based on a Deprotected Poly(N-vinylacetamide)

Using the PVA obtained from deprotected 4,060 kDapoly(N-vinylacetamide)-linear homopolymer from Example 31 a very highmolecular weight hydrophobically modified stimuli responsive polymer wasprepared as follows.

100 g of a 2% solution of deprotected 4,060 kDa poly(N-vinylacetamide)was placed in a glass jar. To the glass jar, 100 g of 4 molar NaOH wasadded to adjust to pH to approximately 13. Next, 20 g of 1-propanol wasadded to the jar as a co-solvent. Finally, 0.58 g of benzyl chloride wasadded and the jar was capped. The reaction was heated at 60° C. for 3hours with vigorous shaking. After 3 hours, the reaction mixture wascooled to room temperature and the product was precipitated with acetoneand subsequently collected. The resulting solids were washed withdeionized water, followed by isopropyl alcohol, and were dried in avacuum oven at 65° C. for 2 days. The dry solids were ground to a finepowder and the final dry mass of polymer collected was 1.44 g. A 2%solution was made by dissolving the dried powder into 1 molar aceticacid and 0.08% HCl. The resulting solution was tested for sensitivity toa multivalent ion stimulus by drop wise addition of 2 molar sodiumphosphate or 0.2 molar sodium citrate to 5 mL samples of 0.5% polymersolutions. Upon addition of the phosphate or citrate ions a whiteprecipitate is observed, thereby indicating that the polymer wasresponsive to a multivalent anion stimulus.

FIG. 6 provides a schematic of the synthesis process described in thisExample.

Example 33 Synthesis of a Stimulus Responsive Vinylamine/VinylbutyletherCopolymer (VA-co-VBE)

In another experiment, a stimulus responsive copolymer was prepared,which had one of the monomeric units included a hydrophobic group.

A stimulus responsive polymer comprised of repeat units containingprimary amines and butylether was synthesized from monomers as follows.90 g of octane, 2.5 g of Span-85 (SIGMA), 16 g of N-vinylformamide(NVF)(ALDRICH, 98%), 5 g of N-butylvinylether (SIGMA), and 30 g ofdeionized water was placed in a 250 mL round bottom flask. The flask wasequipped-with a magnetic stirrer and a N₂ dipstick. The solution wasstirred and purged with N₂ for 1 hour as the temperature was increasedto 55° C.

Initiator solution was prepared by adding 0.10 g of 2,2′Azobis(2-amidinopropane)dihydrochloride (ABAP) (ALDRICH) to 10 mL ofdeionized water and dissolved. The initiator solution was charged to the250 mL round bottom flask containing the reaction solution undernitrogen purged atmosphere. The solution was heated at 55° C. for 1hour, followed by heating at 60° C. for 1 hour, further followed byheating at 70° C., for 1 hour and followed by heating at 80° C. for 1hour, with vigorous stirring with continuous nitrogen purging. Thisresulted in a two phase solution with a viscous gel layer on the bottom.The top layer was decanted and discarded. To the bottom layer, 200 mL ofdeionized water was added and 20 g of 50% NaOH was added with vigorousstirring. The solution was heated at 80° C. for 6 hours. After 6 hours,the solution was removed from heat and allowed to cool to roomtemperature. The volume was increased to 2 L with deionized water. Theproduct was isolated by dropwise addition of 2 molar sodium phosphate,resulting in a large white precipitate. The precipitate was collected bydecanting the supernatant and the precipitate was washed with deionizedwater. The isolated polymer was dissolved in 500 mL deionized water, 10g acetic acid, and 2 g concentrated HCl. The resulting solution wastested for sensitivity to a multivalent ion stimulus by drop wiseaddition of 2 molar sodium phosphate or 0.2 molar sodium citrate to 5 mLsamples of 0.5% polymer solutions. Upon addition of the phosphate orcitrate ions a white precipitate was observed, thereby indicating thatthe polymer was responsive to a multivalent anion stimulus.

FIG. 7 depicts a schematic of the reaction described in this Example.This example demonstrates that copolymers containing an amine or chargedfunctionality copolymerized with a hydrophobic monomer are responsive toa multivalent ion stimulus.

Example 34 Synthesis of Very High Molecular Weight Polyvinylamine (PVA)Stimulus Responsive Polymer from NVF Monomer via Inverse EmulsionPolymerization

A stimulus responsive polymer comprised of repeat units containingprimary amines was synthesized from a monomer as follows. 90 g ofoctane, 2.5 g of Span-85 (SIGMA), 16 g of N-vinylformamide(NVF)(ALDRICH, 98%), and 30 g of deionized water was placed in a 250 mLround bottom flask. The flask was equipped with a magnetic stirrer and aN₂ dipstick. The solution was stirred and purged with N₂ for 1 hour asthe temperature was increased to 55° C. Initiator solution was preparedby adding 0.20 g of 2,2′ Azobis(2-amidinopropane)dihydrochloride (ABAP)(ALDRICH) to 20 mL of deionized water and dissolving. The initiatorsolution was charged to the 250 mL round bottom flask containing thereaction solution under nitrogen purged atmosphere. The solution washeated to 60° C. for 2 hours, followed by heating to 75° C. for 1 hourwith vigorous stirring with continuous nitrogen purging. A two phasesolution results with a viscose gel layer on the-bottom. The lop layeris decanted and discarded. To the bottom layer 500 mL of deionized wateris added and 48 g of 50% NaOH is added with vigorous stirring. Thesolution was heated to 80° C. for 16 hours. After 16 hours the solutionwas removed from heat and allowed to cool to room temperature. Thevolume was increased to 1 L with deionized water. The product wasisolated by drop wise addition of 2 molar sodium phosphate resulting ina large white precipitate. The precipitate was collected by decantingthe supernatant and the precipitate was washed with deionized water andsoaked in isopropyl alcohol for 2 hours and finally washed again withdeionized water. The resulting solid mass was dried in a vacuum oven at65° C. for 3 days. The dried polymer was frozen with liquid nitrogen andground to a fine powder and further dried for 1 day. The resulting massof the dry powder was 21.5 g. A 2% solution was made by dissolving thedried powder into 1 molar acetic acid and 0.08% HCl. The resultingsolution was tested for sensitivity to a multivalent ion stimulus bydrop wise addition of 2 molar sodium phosphate or 0.2 molar sodiumcitrate to 50 mL samples of 1% polymer solutions. Upon addition of thephosphate or citrate ions a white precipitate is observed.

Example 35 Synthesis of a High Molecular Weight Hydrophobically ModifiedPolyvinylamine (PVA) Stimulus Responsive Polymer

Using a solution of Lupamin 9095 (linear polyvinylamine, average MW=340kDa, 20% solids, pH 7-9) obtained from BASF, a hydrophobically modifiedstimulus responsive polymer was produced as follows. 300 g of Lupamin9095 (approximately 60 g of polyvinylamine) was added to a 2 liter glassjar. Next, 40 g of NaOH pellets and 500 mL of deionized water weredissolved and added to the jar. This was followed by the addition of 500mL 1,2-dimethoxyethane (SIGMA) as a co-solvent and the solution wasstirred vigorously until homogenous. Next, 17.66 g benzyl chloride(ACROS ORGANICS, 99%) was added to the reaction jar with stirring. Thesolution was heated to 60° C. for 16 hours with magnetic stirring. Thesolution was subsequently allowed to cool to room temperature andtransferred to a 5 liter beaker. Next, 1500 mL of deionized water wasadded with stirring. The pH was adjusted to 5 with glacial acetic acid.The product was precipitated with slow addition of 250 mL 2 molar sodiumphosphate and the solid was collected and washed with deionized water.

The polymer was further purified by the following method. The solid wasdissolved in 2 liter of 1 molar acetic acid with stirring. The totalvolume was brought to 10 liters with deionized water and the pH isadjusted to 7 with drop wise addition of 50% NaOH. The product wasprecipitated with addition 600 g of 2 molar sodium phosphate. The solidwas isolated via vacuum filtration and washed with deionized water. Theresulting solid mass was dried in a vacuum oven at 65° C. for 3 days.The dried polymer was frozen with liquid nitrogen and ground to a finepowder and further dried for 1 day. The resulting mass of the dry powderwas 46 g. A small sample was dissolved in 1 molar CD₃COOD/D₂O acid and¹H-NMR spectra were obtained, which are depicted in FIG. 8. The ¹H-NMRpeaks were integrated and the amount of benzyl modification wasdetermined to be 18%.

Example 36 200 a Scale Synthesis of a Hydrophobically ModifiedPolyallylamine Based Stimulus Responsive Polymer

In an exemplary experiment described herein, it was demonstrated thatthe polymers described herein could be manufactured on a large scale.

Using a solution of Polyallyamine (PAA, NITTOBO, 150 kD; 40% wt./wt.), ahydrophobically modified stimulus responsive polymer was produced asfollows. 500 g of Polyallyamine (PAA, NITTOBO, 150 kD; 40% wt./Wt.)(approximately 200 g of polyallylamine) was added to a 4 liter glassjar. Next, 80 g of NaOH pellets and 1000 mL of deionized water weredissolved and added to the jar. Followed by the addition of 1000 mL1,2-dimethoxyethane (SIGMA) as a co-solvent and the solution was stirredvigorously until homogenous. Next, 114 g of benzyl chloride (ACROSORGANICS, 99%) was added to the reaction jar. The solution was heated at60° C. for 16 hours with magnetic stirring. The solution was allowed tocool to room temperature and transferred to a 10 liter beaker.

Next, 1000 mL of deionized water was added with stirring and a stickysolid mass precipitated out of solution. The product was furtherprecipitated with slow addition of 200 mL 2 molar sodium phosphate andthe solid was collected and washed with deionized water. The polymer wasfurther purified by the following method. The solid was dissolved in 3liter of 1 molar acetic acid with stirring. The total volume was broughtto 10 liters with deionized water and the pH was adjusted to 7 with dropwise addition of 50% NaOH. The product was precipitated with addition800 g of 2 molar sodium phosphate and the solids was collected andwashed with deionized water. The polymer was even further purified bythe following method. The solid was dissolved in 3 liter of 1 molaracetic acid with stirring. The total volume was brought to 10 literswith deionized water and the pH was adjusted to 7 with drop wiseaddition of 50% NaOH. The product was precipitated with addition 800 gof 2 molar sodium phosphate and the solids was collected and washed withdeionized water. The resulting solid mass was dried in a vacuum oven at65° C. for 3 days. The dried polymer was frozen with liquid nitrogen andground to a fine powder and further dried for 1 day. The resulting massof the dry powder was 250 g. A small sample was dissolved in 1 molarCD₃COOD/D₂O acid and ¹H-NMR spectra were obtained, as depicted in FIG.9. The ¹H-NMR peaks were integrated and the amount of benzylmodification was determined to be 33%.

Example 37 Determination of Flocculation Performance and SupernatantQuality for Increasing Doses of a Stimulus Responsive Polymer vs aCationic Polyelectrolyte in a CHO Cell Culture

In an exemplary experiment described herein, the stimulus responsivepolymers according to the present invention were compared to a knownpolymer, i.e., chitosan, for certain desirable properties.

CHO cell culture was prepared using a method, as described in Example 1:A solution of 2% w/w solution of medium molecular weight chitoson (MMWchitosan) (Sigma-Aldrich) was prepared in 1 molar acetic acid. A 2% w/wsolution of hydrophobically modified polyallylamine based stimulusresponsive (33%-BnPAA) polymer was prepared according to Example #36. 10mL of the CHO cell culture was dispensed into 15 mL conical tubes.Individual polymer doses of 0.0, 0.1, 0.2, 0.03, 0.04, 0.05, 0.06, 0.07.0.08, 0.09, 0.1, 0.14, 0.18, 0.22, and 0.4% w/v were added to eachconical tube containing CHO cell culture for each the LMW chitosan and33%-BnPAA. For the conical tubes containing the 33%-BnPAA only, the pHwas adjusted to 7.2 and a stimulus of 150 mM sodium phosphate wasapplied. All the conical tubes were centrifuged at 3000 RPM for 2minutes and the supernatant was decanted and the turbidity wasdetermined.

Table 5 below and FIG. 10 summarize the results of a representativeexperiment to demonstrate that a non-stimulus responsive polymer (e.g.,chitosan) requires dose optimization for efficient flocculation,whereas, a stimulus responsive polymer according to the presentinvention does not appear to require dose optimization. In other words,in case of a non-stimulus responsive polymer such as chitosan, once anoptimal dose of the polymer is added for efficient flocculation,exceeding that optimal dose results in increased turbidity, which isundesirable. Whereas, in case of stimulus responsive polymers such asthose described herein, the stimulus responsive polymer continues to bean effective flocculant/precipitant regardless of increase in the dose.

TABLE 5 Polymer Flocculant Stimulus Supernatant Dose (w/v)% PolymerFlocculant (y/n) Turbidiy (NTU) 0 MMW Chitosan n 266 0.01 MMW Chitosan n14 0.02 MMW Chitosan n 13 0.03 MMW Chitosan n 28 0.04 MMW Chitosan n 2000.05 MMW Chitosan n 282 0.06 MMW Chitosan n 311 0.07 MMW Chitosan n 3250.08 MMW Chitosan n 353 0.09 MMW Chitosan n 378 0.1 MMW Chitosan n 3790.14 MMW Chitosan n 404 0.18 MMW Chitosan n 409 0.22 MMW Chitosan n 4050.4 MMW Chitosan n 400 0 33% Benzyl modified y 237 Polyallylamine 0.0133% Benzyl modified y 399 Polyallylamine 0.02 33% Benzyl modified y 486Polyallylamine 0.03 33% Benzyl modified y 433 Polyallylamine 0.04 33%Benzyl modified y 293 Polyallylamine 0.05 33% Benzyl modified y 42Polyallylamine 0.06 33% Benzyl modified y 19 Polyallylamine 0.07 33%Benzyl modified y 14 Polyallylamine 0.08 33% Benzyl modified y 16Polyallylamine 0.09 33% Benzyl modified y 21 Polyallylamine 0.1 33%Benzyl modified y 20 Polyallylamine 0.14 33% Benzyl modified y 17Polyallylamine 0.18 33% Benzyl modified y 17 Polyallylamine 0.22 33%Benzyl modified y 12 Polyallylamine 0.4 33% Benzyl modified y 14Polyallylamine

Example 38 Determination of Flocculation Performance and SupernatantQuality with Increasing Doses of a Stimulus Responsive Polymer in thePresence of a Multivalent Anion Stimulus Relative to No Stimulus, in CHOCell Culture

CHO cell culture was prepared using a method, as described in Example 1.A 2% w/w solution of hydrophobically modified polyallylamine basedstimuli responsive (33%-BnPAA) polymer was prepared according to Example#36. Individual polymer doses of 0.0, 0.05, 0.06, 0.07. 0.08, 0.09, 0.1,0.14, 0.18, 0.22, and 0.4% w/v were added to each conical tubecontaining CHO cell culture in triplicates (one for phosphate and onefor citrate and one for no stimuli) for 33%-BnPAA polymer. The pH wasadjusted to 7.2 and a stimulus of 150 mM sodium phosphate or 150 mMsodium citrate was applied or no stimulus was applied. All the conicaltubes were centrifuged at 3000 RPM for 2 minutes and the supernatant wasdecanted and the centrate turbidity was determined. The results from theexperiments described in this example are shown in Table 6 and FIG. 11.

Table 6 and FIG. 11 summarize the results of a representative experimentto demonstrate that a stimulus responsive polymer (e.g., described inExample 36) can operate as a non-stimulus responsive flocculant (similarto Chitosan data in Example 37), requiring polymer dose optimizationwithout a stimulus. However, with a multivalent ion stimulus, such asphosphate or citrate, the stimulus responsive polymer does not requiredose optimization as the centrate turbidity is not effected byincreasing polymer dose.

TABLE 6 Polymer Stimulus Supernatant Flocculant (phosphate/ TurbidiyDose (w/v)% Polymer Flocculant citrate/none) (NTU) 0 33% Benzyl modifiedphosphate 237 Polyallylamine 0.05 33% Benzyl modified phosphate 42Polyallylamine 0.06 33% Benzyl modified phosphate 19 Polyallylamine 0.0733% Benzyl modified phosphate 14 Polyallylamine 0.08 33% Benzyl modifiedphosphate 16 Polyallylamine 0.09 33% Benzyl modified phosphate 21Polyallylamine 0.1 33% Benzyl modified phosphate 20 Polyallylamine 0.1433% Benzyl modified phosphate 17 Polyallylamine 0.18 33% Benzyl modifiedphosphate 17 Polyallylamine 0.22 33% Benzyl modified phosphate 12Polyallylamine 0.4 33% Benzyl modified phosphate 14 Polyallylamine 0.0533% Benzyl modified citrate 50 Polyallylamine 0.06 33% Benzyl modifiedcitrate 29 Polyallylamine 0.07 33% Benzyl modified citrate 27Polyallylamine 0.08 33% Benzyl modified citrate 23 Polyallylamine 0.0933% Benzyl modified citrate 30 Polyallylamine 0.1 33% Benzyl modifiedcitrate 21 Polyallylamine 0.14 33% Benzyl modified citrate 19Polyallylamine 0.18 33% Benzyl modified citrate 24 Polyallylamine 0.2233% Benzyl modified citrate 23 Polyallylamine 0.4 33% Benzyl modifiedcitrate 20 Polyallylamine 0.05 33% Benzyl modified none 64Polyallylamine 0.06 33% Benzyl modified none 33 Polyallylamine 0.07 33%Benzyl modified none 23 Polyallylamine 0.08 33% Benzyl modified none 46Polyallylamine 0.09 33% Benzyl modified none 51 Polyallylamine 0.1 33%Benzyl modified none 55 Polyallylamine 0.14 33% Benzyl modified none 199Polyallylamine 0.18 33% Benzyl modified none 492 Polyallylamine 0.22 33%Benzyl modified none 454 Polyallylamine 0.4 33% Benzyl modified none 455Polyallylamine

Example 39 Determination of Flocculation Performance and SupernatantQuality with Increasing Doses of an Unmodified Stimulus ResponsivePolymer in CHO Cell Culture

In another experiment, the flocculation ability and supernatant qualitywith an unmodified stimulus responsive polymer in a CHO cell culture wasdetermined, as described herein.

CHO cell culture was prepared using a method, as described in Example 1.A 2% w/w solution of unmodified polyallylamine based stimuli responsivepolymer was prepared by dissolving Polyallyamine (PAA, NITTOBO, 150 kD;40% wt./wt.) in 1 molar acetic acid. 10 mL of the CHO cell culture isdispensed into 15 mL conical tubes. Individual polymer doses of 0.0,0.05, 0.06, 0.07. 0.08, 0.09, 0.1, 0.14, 0.18, 0.22, and 0.4% w/w wasadded to each conical tube containing CHO cell culture. The pH wasadjusted to 7.2 and a stimulus of 150 mM sodium phosphate was applied.All the conical tubes were centrifuged at 3000 RPM for 2 minutes and thesupernatant was decanted and the turbidity was determined. The resultsfrom the experiments described in this example are shown in Table 7.

TABLE 7 Polymer Supernatant Flocculant Stimulus Turbidiy Dose (w/v)%Polymer Flocculant (y/n) (NTU) 0.05 umodified 150 kD n 171 Polyallyamine0.06 umodified 150 kD n 426 Polyallyamine 0.07 umodified 150 kD n 673Polyallyamine 0.08 umodified 150 kD n 959 Polyallyamine 0.09 umodified150 kD n 947 Polyallyamine 0.1 umodified 150 kD n >1000 Polyallyamine0.14 umodified 150 kD n >1000 Polyallyamine 0.18 umodified 150 kDn >1000 Polyallyamine 0.22 umodified 150 kD n >1000 Polyallyamine 0.4umodified 150 kD n >1000 Polyallyamine

Example 40 Determination of Flocculation Performance, Settling Time, andSupernatant Quality a Stimuli Responsive Polymer in CHO Cell Culture

CHO cell culture was prepared using a method as described in Example 1.A 10% w/w solution of a hydrophobically modified polyallylamine basedstimuli responsive (33%-BnPAA) polymer was prepared similar to Example#36. 50 mL of CHO cell culture was placed in a 100 mL glass graduatedcylinder in duplicates. In one of the cylinders, the stimulus responsivepolymer was added to a dose of 0.5%, the pH was adjusted to 7 with dropwise addition of 2 molar tris base, the sodium phosphate concentrationwas adjusted to 50 mM by addition of a 2 molar sodium phosphatesolution, and the solution was stirred for 2 minutes. The sodiumphosphate and adjustment was performed in order to precipitate thestimulus responsive polymer and a complex of cells, cell debris,impurities, residual polymer; to flocculate the solids; and to increaseparticle size. After the sodium phosphate addition and pH adjustment,large aggregated particles were observed in the stimulus responsivepolymer treated feed. The other graduated cylinder was stirred for 2minutes and nothing was added. Both of the cylinders were allowed to situndisturbed for 1 hour. At the end of an hour, the supernatant wasaspirated and the turbidity was recorded.

It was observed that the solid phase in the cylinder with the smartpolymer settles faster as well as a defined settling front (sharp solidto liquid phase transition) relative to the solid phase in the untreatedcylinder, which gave an undefined settling front.

The results of one such experiment are summarized in Table 8. Themeasurements with respect to the settling fronts were a rough estimatefor the untreated cylinder because the settling front was largelydispersed and undefined.

TABLE 8 Final Settling Supernatant Time Front (% Turbidity (minutes)Polymer Solids) (NTU) 10 33% Benzyl modified 100 Polyallylamine 20 33%Benzyl modified 100 Polyallylamine 30 33% Benzyl modified 62Polyallylamine 40 33% Benzyl modified 50 Polyallylamine 50 33% Benzylmodified 47 Polyallylamine 60 33% Benzyl modified 42 18 Polyallylamine10 None 100 20 None 100 30 None 95 40 None 93 50 None 88 60 None 84 589

Example 41 Comparison of Clarification Performance Using Polyamines ofDifferent Molecular Weights

A series of polymers with different molecular weights were obtained,modified, and used to flocculate, precipitate, and purify a cellculture. Polymers with primary amine repeat units of molecular weightsof 15 kD, 85 kD, 150 kD, 350 kD, 600-950 kD and 2000-4000 kD wereobtained and/or modified by the following methods.

The 15 kD polyallylamine polymer was obtained from NITTOBO and wasbenzylated (covalently attached benzyl groups and purified) using amethod similar to example 36. The 85 kD benzylated polyvinylaminepolymer was prepared by Example 23. The 150 kD benzylated polyallylaminewas prepared according to Example 36. The 350 kD benzylatedpolyvinylamine polymer was prepared by Example 35. The 950 kDpolyvinylamine polymer backbone is prepared by hydrolyzing Polymin VZ(BASF) with 2 equivalents of base at 80° C. for 8 hours. The unmodified2000-4000 kD PVA was prepared according to example 31. The benzylated2000-4000 kD polymer was prepared according to Example 32. The polymerswere used to flocculate, precipitate, and purify a CHO DG44 cell cultureof approximately 12×10⁶ cells/mL and a harvest cell viability of <50%.The flocculation was performed at polymer doses of 0.2% and 0.4% w/w. Asolution stimulus of 50 mM sodium phosphate and pH adjustment to 7 with2 molar tris base was applied.

Observations about the flock size were recorded and are denoted as +being small flocks and +++++ being very large aggregates. The vialsdenoted +++++ were more of a complete zone separation than a suspensionof aggregates. It is also noted that the larger aggregates/particlessettle faster with a sharper solid liquid interface. Results are shownin Table 9, which demonstrates the results for flocculation usingpolyamines of different molecular weights.

TABLE 9 Molecular Weight Polymer Dose Aggregate/Particle (kD) (w/w) Size15 0.2 + 85 0.2 + 150 0.2 ++ 350 0.2 +++ 600-900 0.2 +++ 2000-4000 0.2++++ 15 0.4 + 85 0.4 + 150 0.4 +++ 350 0.4 ++++ 600-900 0.4 ++++2000-4000 0.4 +++++

Example 42 Comparison of Clarification Performance Using Polyamines ofDifferent Hydrophobic Modifications

CHO cell culture was prepared using a method, as described in Example 1.Samples of polymers described in Examples 31, 32, 33 and 35 were addedto the cell culture as described in Example 38 with the followingexceptions: The polymer dose was between 0.1 wt % and 0.6 wt % asdescribed in Table 10. The initial cell culture turbidity was ˜900 NTUand the centrate without polymer treatment had a turbidity of 212 NTU.The pH was adjusted to 7.2 and a stimulus of 50 mM sodium phosphate wasapplied. All the conical tubes were centrifuged at 3000 RPM for 2minutes and the supernatant was decanted and the centrate turbidity wasdetermined. The results from the experiments described in this exampleare shown in Table 10, which demonstrates the performance of differenthydrophobic modifications at different polymer doses.

Table 10 demonstrates that by changing the nature of the hydrophobicgroup and/or polymer molecular weight, the response to stimulus andresulting centrate turbidity can be varied.

TABLE 10 Centrate Polymer Dose Turbidity Polymer (w/w) (NTU) Untreated 0212 (No Polymer) Example 31 0.2 12 Example 31 0.6 76 Example 32 0.2 92Example 32 0.6 26 Example 33 0.2 17 Example 33 0.6 61 Example 35 0.1 19Example 35 0.2 12 Example 35 0.4 36 Example 35 0.6 32

Example 43 Effect of Reduced Turbidity Obtained with Stimulus ResponsivePolymers on Downstream Filtration

In order to assess the affect of the stimulus responsive polymer onsubsequent centrifugation and depth filtration steps, the followingprocedure was followed. DG44 chinese Hamster Ovary (CHO) cell lineexpressing PTG1 antibody were grown in a 10 L bioreactor (NEW BRUNSWICKSCIENTIFIC) to a density of about 15×106 cells/mL and harvested at <50%viability. A 10% w/w solution of a hydrophobically modifiedpolyallylamine based stimuli responsive (33%-BnPAA) polymer is preparedsimilar to Example 36.

One fraction of the DG44 CHO cell culture is treated with 0.2% of thestimulus responsive polymer 33%-BnPAA, while another fraction of theDG44 CHO cell culture is not treated. To the stimulus responsive polymertreated cell culture, the sodium phosphate concentration is brought to50 mM by drop wise addition of 2 molar sodium phosphate and the pH isadjusted to 7.2 by drop wise addition of 2 molar tris base. The sodiumphosphate and pH adjustment is performed in order to precipitate thestimulus responsive polymer and a complex of cells, cell debris,impurities, residual polymer, and to flocculate the solids, and increaseparticle size. After the sodium phosphate addition and pH adjustment,large flocked particles are observed in the stimuus responsive polymertreated feed. Both fractions were centrifuged for 5 minutes at 3000 RPMand the supernatants were decanted and the turbidity was determined. Thecentrate turbidity for the 33%-BnPAA polymer treated feed was determinedto be 10 NTU while the centrate turbidity for the untreated feed wasdetermined to be 60 NTU.

Depth filter throughput for each feed was determined by the followingmethod. A X0HC Millistak+® Pod Disposable Depth Filter (MILLIPORE) witha surface area of 23 cm² used for each feed. The depth filters wereequipped with a peristaltic pump and an in-line pressure sensor-Thefilters were flushed with deionized water according to instructions andthe feed was pumped through at 100 LMH and the filtrates were pooled.The pooled turbidity for the 33%-BnPAA polymer treated feed filtrate was6 NTU and the pooled turbidity for the untreated feed filtrate was 9NTU. The filter throughput for the 33%-BnPAA polymer treated feed was1304 L/m2 at 10 psi, at which time the experiment was stopped because offeed limitations. The filter throughput for the untreated feed was 206L/m2 at 20 psi, at which time the experiment was stopped because ofpressure limitations.

Example 44 Purification of a Model Protein Stream with a StimulusResponsive Polymer Followed by Capture with a Affinity Resin

In order to better assess the affect of the stimulus responsive polymeron subsequent purification steps, a model feed was prepared and thefollowing procedure was followed. CHO cell culture is prepared using amethod similar to Example 1. Initial Feed HCP level ˜210,000ppm. A 10%w/w solution of a hydrophobically modified polyallylamine based stimuliresponsive (33%-BnPAA) polymer is prepared similar to Example 36. Onefraction of the CHO cell culture is treated with 0.1% of the stimuliresponsive polymer 33%-BnPAA, while another fraction of the CHO cellculture is treated with 0.4% of the stimuli responsive polymer33%-BnPAA, and a third fraction of the CHO cell culture is not treated.To the stimuli responsive polymer treated cell cultures the sodiumphosphate concentration is brought 50 mM by drop wise addition of 2molar sodium phosphate and the pH is adjusted to 7.2 by drop wiseaddition of 2 molar tris base. The sodium phosphate and pH adjustment isperformed in order to precipitate the stimuli responsive polymer and acomplex of cells, cell debris, impurities, residual polymer, and toflocculate the solids, and increase particle size. After the sodiumphosphate, addition and pH adjustment large flocked particles areobserved in the stimuli responsive polymer treated feeds. Each fractionof the cell culture is centrifuged in a lab scale bucket centrifuge for5-minutes at 3000 RPM. The turbidty of each centrate is recorded andreported in Table 11. The centrates are filtered through a 0.2μmDurapore® filter.

The filtrate pools were purified through a three step chromatographybased purification consisting of protein A affinity chromatography(ProSep Ultra Plus®), bind-and-elute cation exchange chromatography(ProRes S®), and membrane adsorber anion exchange chromatography inflow-though mode (ChromaSorb®). The purification was performed on achromatography workstation according to the method in Table 12. Thepools for each step were analyzed for host cell protein (CHOP) by ELISA,leached Protein A (L ProA) by ELISA, residual DNA by PicoGreen® assay,turbidity, aggregated protein (AGG) percent by size exclusion HPLC, andprotein concentration by UV absorption.

TABLE 11 Mab CHOP Feed Centrate Recovery Removal DNA Treatment (NTU) (%)(%) (ug/mL) Centrifuge 127 100 0 49 Only 0.1% 7.3 90 50 <LOQ Bn25PAA0.4% 9.2 78 50 <LOQ Bn25PAA Protein A Pools Virus Inactivation, StepFeed low pH hold Yield [CHOP] L ProA DNA Treatment Pool (NTU) (%) (ppm)AGG % (ppm) (ug/mL) Centrifuge 45 99 2135 3 >25 0.84 Only 0.1% 2.4 99507 2.9 12 <LOQ Bn25PAA 0.4% 2 100 267 3 >25 <LOQ Bn25PAA Centrifuge 87684 2.3 <LOQ <LOQ Only 0.1% 91 282 2.4 <LOQ <LOQ Bn25PAA 0.4% 86 133 2.4<LOQ <LOQ Bn25PAA ChromaSorb Pools Feed Step Yield [CHOP] DNA Treatment(%) (ppm) L ProA (ug/mL) Centrifuge 93 1.0 or <LOQ <LOQ Only <LOQ 0.1%96 0.7 or <LOQ <LOQ Bn25PAA <LOQ 0.4% 95 0.7 or <LOQ <LOQ Bn25PAA <LOQ

TABLE 12 Protein A Column Dimensions 0.66 × 14 cm Method Duration(Column Residence Step Buffer Volumes) Time EQ Phospahte buffered saline5 3 Load 0.9 mg/mL Clarified feedstock 214 mL 4 Wash Phospahte bufferedsaline 9 3 Elution 50 mM Acetic acid, pH 3.0 6 3 Acid Strip 150 mMPhosphoric acid 3 3 EQ Phospahte buffered saline 5 3 Cation ExchangeColumn Dimensions 0.66 × 14 cm Method Duration (Column Residence StepBuffer Volumes) Time EQ 50 mM Acetic acid, 25 mM NaCl, pH 5.0 3 3 LoadProtein A elution pool adjusted to pH 5 load density = 4 with 50 mMAcetic acid, 25 mM NaCl, 40 mg/mL pH 5.0 Wash 50 mM Acetic acid, 25 mMNaCl, pH 5.0 3 3 Elution 50 mM Acetic acid, 125 mM NaCl, pH 5.0 6 3 NaClStrip 50 mM Acetic acid, 500 mM NaCl, pH 5.0 3 3 NaOH Cleaning 0.5M NaOH3 3 EQ 50 mM Acetic acid, 25 mM NaCl, pH 5.0 15  3 Anion ExchangeChromaSorb 0.08 mL Method Duration (Column Flow Rate Step BufferVolumes) (mL/minute) EQ 50 mM Tris pH 7.4 300 1 Load Cation exchangepool adjusted with Load Density = 1 50 mM tris pH 7.4 2.5 kg/L

Example 45 Purification of a Model Protein Stream with a StimulusResponsive Polymer Followed by Capture with a Cation Exchange Resin

In order to better assess the affect of the stimulus responsive polymeron subsequent purification steps, the following procedure was followed.DG44 Chinese Hamster Ovary (CHO) cell line expressing PTG1 antibody weregrown in a 10 L bioreactor (New Brunswick Scientific) to a density ofabout 15×106 cells/mL and harvested at <50% viability with an HCP levelof ˜142000 ppm.

A 10% w/w solution of a hydrophobically modified polyallylamine basedstimuli responsive (33%-BnPAA) polymer was prepared similar to Example#36. One fraction of the CHO cell culture was treated with 0.1% of thestimuli responsive polymer 33%-BnPAA, while another fraction of the CHOcell culture was treated with 0.4% of the stimuli responsive polymer33%-BnPAA, and a third fraction of the CHO cell culture was not treated.To the stimuli responsive polymer treated cell cultures, sodiumphosphate concentration was brought to 50 mM by drop wise addition of 2molar sodium phosphate and the pH was adjusted to 7.2 by drop wiseaddition of 2 molar tris base. The sodium phosphate and pH adjustmentwas performed in order to precipitate the stimuli responsive polymer anda complex of cells, cell debris, impurities, residual polymer, and toflocculate the solids, and increase particle size. After the sodiumphosphate addition and pH adjustment large aggregated particles areobserved in the stimuli responsive polymer treated feeds. Each fractionof the cell culture is centrifuged in a lab scale bucket centrifuge for5 minutes at 3000 RPM. The turbidty of each centrate is recorded andreported in Table 13. The centrates were filtered through a 0.2 μmDurapore® filter.

The filtrate pools were purified through a two step chromatography basedpurification consisting of bind-and-elute cation exchange chromatography(ProRes S®), and membrane adsorber anion exchange chromatography inflow-though mode (ChromaSorb®). The purification was performed on achromatography workstation according to the method in Table 12. Thepools for each step were analyzed for host cell protein (CHOP) by ELISA,leached Protein A (L ProA) by ELISA, residual DNA by PicoGreen® assay,turbidity, aggregated protein (AGG) percent by size exclusion HPLC, andprotein concentration by UV absorption.

TABLE 13 Centrate Mab Recovery CHOP DNA Feed Treatment (NTU) (%) Removal(%) (ug/mL) untreated >1000 100 0 11.9 0.1% Polymer 2 92 56 <LOQ 0.4%Polymer 12 90 65 <LOQ CEX Pools StepYield DNA Feed Treatment (%) [CHOP](ppm) Agg (%) (ug/mL) 0.1% Polymer 80 897 <0.1 <LOQ 0.4% Polymer 84 500<0.1 <LOQ ChromaSorb Pools Step Yield DNA Feed Treatment (%) [CHOP](ppm) (ug/mL) 0.1% Polymer 96 270 <LOQ 0.4% Polymer 97 225 <LOQ

Example 46 Polyethylene Membrane Surface Modified with Phosphoric Acid2-Hydroxyethylmethacrylate (PAHEMA)

In another experiment, a membrane was modified to incorporate amultivalent ion stimulus, where the membrane can be used for removal ofresidual stimulus responsive polymer.

A 16% aqueous mixture of PAHEMA was prepared according to the followingrecipe: 16 g of PAHEMA (Aldrich #695890, 75% PAHEMA and 25% BisHEMPA),0.2 g of Irgacure 2959, 93.8 g of water. A polyethylene membrane (0.65um, UPDP MILLIPORE) was prewetted with methanol and exchanged into waterand treated with the PAHEMA formulation. The sample was exposed to UVlight, washed with methanol and water, and dried. The weight added tothe membrane by this surface modification was 4.4%. The infraredspectrum of the membrane displayed a strong methacrylate carbonylabsorption. Staining the membrane with methylene blue (a positivelycharged dye) gave a deep blue color with a cyan optical density of 1.43.

Example 47 Hydrophilic Polyethylene Membrane Surface Modified withPhosphoric Acid 2-hydroxyethylmethacrylate (PAHEMA)

A 16% aqueous mixture of PAHEMA was prepared according to the followingrecipe: 16 g of PAHEMA (Aldrich #695890, 75% PAHEMA and 25% BisHEMPA),0.2 g of Irgacure 2959, 93.8 g of water. A hydrophiloic membrane (0.65um, MPLC MILLIPORE) was directly contacted with the PAHEMA solution. UVexposure and washing as above gave a membrane with a 7.6% add on. Theinfrared spectrum of the membrane displayed a strong methacrylatecarbonyl absorption. Staining the membrane with methylene blue (apositively charged dye) gave a deep blue color with a cyan opticaldensity of 1.45.

Example 48 Hydrophilic Polymethacrylate Resin Modified with PhosphoricAcid 2-hydroxyethylmethacrylate (PAHEMA)

In another experiment, a resin was modified to include a stimulus, wherethe modified resin could then be used for removing the residual polymer.

A solution is prepared with the following composition: 60 ml allylglycidyl ether(AGE), 110 g 4M NaOH, 12 g sodium sulfate. To thissolution 60 ml of ToyoPearl65 C media is added and the mixture is placedin a rotating hybridizer at 50 C for 16 hours. The media are separatedand washed by standard procedures. The PAHEMA grafting solution isprepared as follows: 1.0 g PAHEMA, 0.06 g ammonium persulfate, 9.0 gwater. To this solution is added 5 ml of the AGE modified ToyoPearlmedia. The mixture is placed in a hybridizer at 80 C for 16 hours.Separating and washing by standard procedures gives the PAHEMA modifiedresin. This product stains dark blue when treated with a 0.01% aqueoussolution of methylene blue which is a positively charged dye.

Example 49 Stimulus Responsive Polymer Binding with an HydrophilicPolyethylene Membrane Surface Modified with Phosphoric Acid2-hydroxyethylmethacrylate (PAHEMA)

The PAHEMA modified MPLC membrane (Example 47) was used to capturepolyallylamine (PAA) polymer from 3 solutions containing 100, 10, and 1ppm PAA respectively using the following experimental procedure. Amembrane disk 20 mm processed diameter in 15 cc cell holder. PAAsolutions processed at 1.5 cc/sec. The membrane was washed in and out ofcell holder with 100 ml, PBS buffer. Processed membrane stained withPonceau S.

Ponceau S is a negatively charged dye that absorbs strongly topositively charged surfaces. When PAA is absorbed by the PAHEMA modifiedmembrane, the surface is converted from a negative to positive charge.This conversion is easily observed by staining the processed solutionwith Ponceau S. A good measure of degree of staining with Ponceau S isthe magenta optical density as measured with a Macbeth Densitometer. Themagenta optical density values for various processed membranes (i.e.loaded with PAA) on both the upstream and downstream sides of themembrane are shown in Table 14.

As can be seen for the 1 ppm case, all of the PAA is captured on theupstream side of the membrane. For the 15 mL of processed solution, thiscorresponds to 4.8 micrograms of PAA/cm2 of membrane surface.

TABLE 14 PAA Loading Upstream (Topside) Downstream (Bottomside) (PAAprocessed) Magenta Optical Density Magenta Optical Density Unprocessed 00 100 ppm  0.77 0.77 10 ppm 0.75 0.70  1 ppm 0.60 0

Accordingly, based on the results from this Example, it may be concludedthat the modified membranes and resins described herein may be used forreducing the level of residual polymer or completely removing theresidual polymer.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments in this inventionand should not be construed to limit its scope. The skilled artisanreadily recognizes that many other embodiments are encompassed by thisinvention. All publications and inventions are incorporated by referencein their entirety. To the extent that the material incorporated byreference contradicts or is inconsistent with the present specification,the present specification will supercede any such material. The citationof any references herein is not an admission that such references areprior art to the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, cell culture, treatment conditions, and so forth used inthe specification, including claims, are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessotherwise indicated to the contrary, the numerical parameters areapproximations and may vary depending upon the desired properties soughtto be obtained by the present invention. Unless otherwise indicated, theterm “at least” preceding a series of elements is to be understood torefer to every element in the series. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with a true scope and spirit of the invention beingindicated by the following claims.

1. A stimulus responsive polymer comprising a polyelectrolyte backbonecomprising one or more hydrophobic groups attached to the backbone,wherein the polymer is capable of binding and precipitating abiomolecule of interest in a sample upon addition of a stimulus.
 2. Thestimulus responsive polymer of claim 1, wherein the polyelectrolytebackbone comprises at least two monomeric units.
 3. The stimulusresponsive polymer of claim 2, wherein the polyelectrolyte backbonecomprises at least three monomeric units.
 4. The stimulus responsivepolymer of claim 2, wherein each monomeric unit in the polyelectrolytebackbone comprises a charge.
 5. The stimulus responsive polymer of claim2, wherein at least 50% of the monomeric units comprise a charge.
 6. Thestimulus responsive polymer of claim 1, wherein the biomolecule ofinterest is a therapeutic polypeptide.
 7. The stimulus responsivepolymer of claim 1, wherein the biomolecule of interest is an impuritypresent in a sample along with a therapeutic polypeptide.
 8. Thestimulus responsive polymer of claim 7, wherein the impurity is selectedfrom the group consisting of host cell protein, endotoxin, DNA, RNA,viruses, lipids, whole cells and cellular debris.
 9. The stimulusresponsive polymer of claim 1, wherein the stimulus is a multivalention.
 10. The stimulus responsive polymer of claim 9, wherein themultivalent ion is phosphate or citrate.
 11. The stimulus responsivepolymer of claim 7, wherein the therapeutic polypeptide is an antibody.12. The stimulus responsive polymer of claim 11 wherein the antibody isa monoclonal antibody.
 13. A stimulus responsive polymer selected fromthe group consisting of polyallylamine, polyallylamine modified with abenzyl group, polyvinylamine and polyvinylamine modified with a benzylgroup, wherein the stimulus is phosphate or citrate.
 14. A method ofseparating a target molecule from one or more impurities in a sample,wherein the method comprises the steps of: (a) providing a samplecomprising a target molecule and one or more impurities; (b) contactingthe sample with a stimulus responsive polymer according to claim 1,thereby to form a complex of polymer and the one or more impurities; and(c) adding a stimulus to the sample, thereby to precipitate the complexout of solution, thereby to separate the target molecule from one ormore impurities.
 15. The method of claim 14, wherein the method furthercomprises one or more filtration steps.
 16. The method of claim 14,wherein the method further comprises one or more chromatography step.17. The method of claim 14, wherein the stimulus is a multivalent ion.18. The method of claim 17, wherein the multivalent ion is phosphate orcitrate.
 19. The method of claim 14, wherein the polyelectrolytebackbone comprises at least two monomeric units, at least 50% of theunits comprising a charge.
 20. The method of claim 14, wherein thepolyelectrolyte backbone comprises at least three monomeric units, atleast 50% of the units comprising a charge.
 21. The stimulus responsivepolymer comprising the following structure:

wherein the polymer comprises a carbon containing polyelectrolytebackbone; R₁ and R₂ are charged groups which form a part of thebackbone; R3 is a hydrophobic group attached to a charged group in thebackbone; and n is the number of monomeric units in the polymer, whereinn is equal to or greater than
 2. 22. The stimulus responsive polymercomprising the following structure:

Wherein the polymer comprises a carbon containing polyelectrolytebackbone; R₁, R₂ and R₃ are charged groups which form a part of thebackbone; R₄ is a hydrophobic group attached to a charged group in thebackbone; R₅ is a group possessing a charge opposite of the charge foundin R₁, R₂ or R₃; and n is the number of monomeric units in the polymer,wherein n is equal to or greater than
 2. 23. A method of separating anantibody from one or more impurities in a sample using a stimulusresponsive polymer according to claim 1, while minimizing residualamounts of polymer, the method comprising the steps of: (a) providing asample comprising an antibody and one or more impurities; (b) contactingthe sample with a polymer according to claim 1 under a first set ofconditions suitable for the polymer to bind one or more impurities,thereby to form a first complex of polymer and one or more impurities,wherein the first set of conditions comprise adjusting pH or saltconcentration of the sample before, during or after the addition of thepolymer; (c) precipitating the first complex from the sample under asecond set of conditions; (d) contacting the sample with a stimuluscomprising a multivalent ion, thereby to form a second complex ofresidual polymer and multivalent ion; and (e) precipitating the secondcomplex; thereby to separate the antibody from one or more impurities inthe sample while reducing the amount of residual polymer in the sample.24. The method of claim 23, wherein the antibody is a monoclonalantibody.
 25. The method of claim 23, wherein the one or more impuritiesis selected from the group consisting of host cell protein, DNA, RNA,viruses, antibody aggregates and cell culture additives.
 26. The methodof claim 23, wherein the sample comprises a feedstock.
 27. The method ofclaim 23, wherein the sample comprises cell culture media into which anantibody is secreted.
 28. The method of claim 23, further comprising oneor more chromatography steps.
 29. The method of claim 23, furthercomprising one or more filtration steps.
 30. The polymer of claim 1,wherein the polyelectrolyte backbone comprises a polyamine backbone. 31.The polymer of claim 1, wherein the hydrophobic group is a phenyl group.32. A stimulus responsive polymer useful for purifying a biomolecule ofinterest from a sample, the polymer comprising the following structure:

wherein the polymer comprises a carbon containing polyelectrolytebackbone; R₁, R₂ are amines form a part of the backbone; R₃ is ahydrophobic modifying group present on 20% to 75% of the amine groupsand consists of a phenyl group or derivative thereof; and n is thenumber of monomeric units in the polymer, wherein n is equal to orgreater than
 2. 33. A stimulus responsive polymer for separating atarget molecule from one or more impurities comprising the followingformula:

wherein the polymer comprises a carbon containing polyelectrolytebackbone; R₁, R₂ and R₃ are amine groups which form a part of thebackbone; R₄ is a phenyl group which is attached to 5% to 75% of the R₁,R₂ or R₃ groups; R₅ is a cation exchange group modifying 5% to 50% ofthe R_(I), R₂ or R₃ groups; and n is the number of monomeric units inthe polymer, wherein n is equal to or greater than
 2. 34. A method ofseparating an antibody from one or more impurities in a sample using astimulus responsive polymer, while minimizing residual amounts ofpolymer, the method comprising the steps of: (a) providing a samplecomprising an antibody and one or more impurities; (b) contacting thesample with a stimulus responsive polymer under a first set ofconditions suitable for the polymer to bind one or more impurities,thereby to form a first complex of polymer and one or more impurities,wherein the first set of conditions comprise adjusting pH or saltconcentration of the sample before, during or after the addition of thepolymer; (c) flocculating the first complex from the sample under afirst set of conditions; (d) removing the flocculated solids; (e)contacting the sample with a multivalent ion, thereby to form a secondcomplex of residual polymer and multivalent ion; and (f) precipitatingthe second complex thereby to separate the antibody from one or moreimpurities in the sample while reducing the amount of residual polymerin the sample.
 35. A method of separating a target molecule from one ormore impurities in a sample, wherein the method comprises the steps of:(a) providing a sample comprising a target molecule and one or moreimpurities; (b) contacting the sample with a multivalent ion responsivepolymer, wherein a multivalent ion is added before, during or afteraddition of the polymer, thereby to form a complex of polymer and theone or more impurities; (c) precipitating the complex out of solution,thereby to separate the target molecule from one or more impurities. 36.The method of claim 35, wherein the multivalent ion stimulus isphosphate or citrate.
 37. The method of claim 35, wherein the stimulusresponsive polymer is a copolymer.
 38. The method of claim 35, furthercomprising the step of removing a residual amount of polymer after step(c), wherein the step comprises the use of a membrane or resin modifiedwith a multivalent ion.
 39. The method of claim 38, wherein themultivalent ion is phosphate.